BR112020020073A2 - T CELLS THAT EXPRESS A RECOMBINANT RECEIVER, RELATED POLINUCLEOTIDS AND METHODS - Google Patents

Abstract

t cells expressing a recombinant receptor, related polynucleotides and methods. the present invention relates to methods for modifying immune cells, cell compositions containing modified immune cells, kits and articles of manufacture for targeting a nucleic acid sequence encoding a part of a recombinant receptor, for example, a cell receptor recombinant t (tcr), to a particular genomic locus, and / or to modulate gene expression at the genomic locus, and applications thereof in connection with cancer immunotherapy, such as adoptive transfer of modified t cells. in some respects, the nucleic acid sequence integrates in structure into the locus of a receptor that encodes the gene, and in some respects, results in expression of the total recombinant receptor.

Description

Invention Patent Descriptive Report for "T CELLS THAT EXPRESS A RECOMBINANT RECEIVER, RELATED POLYNUCLEOTIDS AND METHODS". Cross Reference to Related Orders

[001] This order claims priority of North American provisional order No. 62 / 653,553, filed on April 5, 2018, entitled "T CELLS THAT EXPRESS A RECOMMENDING, RELATED POLYNUCLEOTIDE RECEIVER AND METHODS." the content of which is incorporated in this document by reference in its entirety. Sequence Listing Reference Incorporation

[002] The present application is being deposited together with the Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 735042015540SeqList.txt, created on April 3, 2019, which is 181 kilobytes long. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety. Field

[003] The present invention relates to methods of modifying immune cells, cell compositions containing modified immune cells, kits and articles of manufacture which aim at a nucleic acid sequence that encodes a part of a recombinant receptor for a genomic locus particular and / or to modulate the expression of the gene in the genomic locus, and applications of it in connection with cancer immunotherapy that includes adoptive transfer of modified T cells. In some respects, the nucleic acid sequence integrates in structure into the locus of a receptor that encodes the gene, and in some respects, results in expression of the total recombinant receptor. Background

[004] Adoptive cell therapies using recombinantly expressed T cell receptors (TCRs) or other antigen receptors (for example, chimeric antigen receptors (CARs)) to recognize tumor antigens represent an attractive therapeutic modality for the treatment of cancers and other diseases. Expression and function of recombinant TCRs or other antigen receptors may be limited and / or heterogeneous in a cell population. Improved strategies are needed to obtain high and / or homogeneous levels of expression and function of recombinant receptors. These strategies can facilitate the generation of cells exhibiting desired levels of expression and / or properties for use in adoptive immunotherapy, for example, in the treatment of cancer, infectious diseases and autoimmune diseases. summary

[005] Genetically modified immune cells expressing a recombinant receptor such as a recombinant T cell receptor (TCR) are provided herein, which are modified by targeted integration of nucleic acid sequences encoding the recombinant receptor. In some respects, the modified cells comprise a modified modified T cell receptor (TRAC) alpha locus and / or a modified modified T cell receptor (TRBC) constant locus. In some respects, the modified TRAC and / or TRBC locus contains nucleic acid sequences encoding a recombinant TCR or a part or a strand thereof. In some respects, the genetically modified cells provided contain TRAC and / or modified TRBC locus that contain a fusion of a transgene sequence that encodes a part of a matching TCR, and the endogenous open reading frame of the gene that co-complicates. a constant domain of the TCR. In some respects, methods for generating a modified T cell are also provided,

related cell compositions, nucleic acids and kits for use in generating the modified cells described in this document.

[006] A genetically modified T cell containing a modified modified T cell receptor (TRAC) alpha locus is provided herein. In some embodiments, a genetically modified T cell containing a modified T cell receptor alpha locus (TRAC) is provided herein, said modified TRAC locus containing a nucleic acid encoding a recombinant TCR or part thereof. the said recombinant TCR or part thereof containing: (i) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), and (ii) a TCR chain beta (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoding TCRβ and the Vα domain, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, wherein the open reading frame encodes at least a part of the recombinant TCR Cα domain.

[007] Genetically modified T cells containing a modified T cell receptor alpha (TRAC) constant locus are provided herein. In some respects, the modified TRAC locus includes a nucleic acid encoding a recombinant TCR or part thereof, said recombinant TCR or part thereof: (i) an alpha TCR chain (TCRα) comprising an alpha domain variable (Vα) and an alpha constant domain (Cα), and (ii) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), where the nucleic acid sequence comprises (a) a transgene sequence encoding the TCRβ and the Vα domain, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, in which the

open reading frame encodes at least part of the Cα domain of the recombinant TCR. In some of any such modalities, a part of Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, where said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, said one or more modifications introduce a or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more non-native cysteines .

[008] Also provided are genetically modified T cells that contain a modified alpha T cell receptor (TRAC) constant locus that includes a nucleic acid encoding a recombinant TCR or part of it, said recombinant TCR or part thereof comprising: (i) an alpha TCR chain (TCRα) comprising a variable alpha domain (Vα) and a constant alpha domain (Cα), and (ii) a beta TCR chain (TCRβ) comprising one of - beta variable domain (Vβ) and a beta constant domain (Cβ), in which the nucleic acid sequence comprises (a) a transgenic sequence encoding TCRβ and the Vα domain, and (b) and a structure of open reading of the endogenous TRAC locus or a partial sequence thereof, where the open reading frame encodes at least a part of the recombinant TCR Cα domain, where the transgene sequence comprises one or more heterologous or regulatory control elements comprising a heterologous promoter, operably linked to control TCR expression when expressed from a cell introduced with the genetically modified T cell.

[009] In some embodiments, the transgene sequence is or has been integrated through homology-directed repair (HDR). In some embodiments, the modified TRAC locus contains a structure fusion of (i) a transgene sequence and (ii) and an open reading frame or a partial sequence of the same as the endogenous TRAC locus. In some of any such modalities, the transgene sequence is structured with one or more exons from the open reading frame or partial sequence from the endogenous TRAC locus.

[0010] In some embodiments, the transgene sequence does not contain a sequence that encodes a 3 'untranslated region (RTU 3') or an intron. In some embodiments, the open reading frame or a partial sequence thereof contains a 3 'RTU from the endogenous TRAC locus. In some embodiments, a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, where the said other part of Cα is me - nor than the natural size of a native Cα.

[0011] In some modalities, the open reading structure or the partial sequence of the same contains at least one intron and at least one exon of the endogenous TRAC locus. In some embodiments, the transgene sequence is structured with one or more exons from the open reading frame or partial sequence from the endogenous TRAC locus.

[0012] In some of any such modalities, the other part of Cα is encoded by a nucleotide sequence that comprises less than four exons, less than three exons, less than two exons, one exon, or less than than a full exon of the open reading frame of the TRAC locus. In some of any such modalities, the other part of Cα is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs of length. In some of these modalities, the other part of the Cα is encoded by an exon 1 part of the TRAC locus, where the exon 1 part is smaller than the natural exon 1 size of the open reading frame of the TRAC locus.

[0013] In some embodiments, the transgene sequence is or has been integrated downstream of the majority of 5 'nucleotide of exon 1 and the amount of the majority of 3' nucleotide of exon 1 of the open reading frame of the endogenous TRAC locus. In some embodiments, said at least a part of Cα is encoded by at least exons 2 to 4 of the open reading frame of the endogenous TRAC locus or at least a part of exon 1 and exons 2 to 4 of the open reading frame of the locus Endogenous TRAC. In some embodiments, at least one Cα portion is encoded by less than the total length of exon 1 of the open reading frame of the endogenous TRAC locus.

[0014] In some embodiments, the encoded TCRα chain is able to dimerize with a TCRβ chain.

[0015] In some modalities, the encoded Cα contains the selected sequence from any of SEQ ID NOS: 14, 15, 19, or 24, or a sequence that displays at least 85%, 86%, 87%, 88 %, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS: 14, 15 , 19, or 24, or a partial sequence of them.

[0016] In some of any of the modalities provided in this document, the coded Cα comprises the sequence selected from any of SEQ ID NOS: 19, 24 and 243 to 252, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more

sequence sequence with any one of SEQ ID NOS: 19, 24 and 243 to 252, or a partial sequence thereof.

[0017] In some modalities, in which another part of Cα is encoded by the transgene sequence. In some embodiments, the other part of Cα and / or said at least part of Cα is encoded by a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of the same or a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof, or a partial sequence thereof. In some embodiments, the other part of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs in length. In some embodiments, the other part of Cα is encoded by a nucleotide sequence that contains less than four exons, less than three exons, less than two exons, one exon, or less than one full exon of the open reading structure of the TRAC locus. In some embodiments, the other part of Cα is encoded by an exon 1 part of the TRAC locus, where the exon 1 part is smaller than the natural exon 1 size of the open reading structure of the TRAC locus. In some embodiments, the other part of Cα is encoded by an exon 1 part of the TRAC locus, where the exon part 1 contains a 5 'portion of exon 1. In some embodiments, the other part of Cα contains an sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 142, or a partial sequence thereof.

[0018] In some embodiments, the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) contains one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids in compared to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0019] In some of any modalities, the Cα and / or Cβ of the recombinant TCR comprises one or more non-native cysteines. In some of any embodiments, the introduction of one or more cysteine residues comprises replacing a non-cysteine residue with a cysteine residue. In some of any modalities, a coded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO: 20. In some of any modalities, the coded Cα comprises the selected sequence of any of the SEQ ID NOS: 248-252, or a partial sequence thereof.

[0020] In any of the modalities provided, the modified T cell can also contain a genetic disruption in a TRBC locus. In some embodiments, the modified T cell also contains a genetic disruption at a TRBC1 locus and / or a TRBC2 locus.

[0021] In some embodiments, the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) contains one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids in comparison to a region

Native Cα and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0022] A genetically modified T cell containing a modified modified T cell receptor (TRBC) beta locus is provided herein. In some embodiments, a genetically modified T cell containing a modified T cell receptor beta (TRBC) constant locus is provided herein, said modified TRBC locus containing a nucleic acid encoding a recombinant TCR or part thereof. therefor, said recombinant TCR or part thereof: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), and (ii) a TCR chain alpha (TCRα) comprising a variable alpha domain (Vα) and a constant alpha domain (Cα), wherein the nucleic acid sequence comprises (a) a transgene sequence encoding TCRα and the Vβ domain, and ( b) and an open reading frame of the endogenous TRBC locus or a partial sequence thereof, in which the open reading frame coexists at least part of the recombinant TCR Cβ domain.

[0023] Genetically modified T cells containing a modified modified T cell receptor (TRBC) beta locus are also provided herein. In some of any such modalities, the modified TRBC locus comprising a nucleic acid encoding a recombinant TCR or part thereof, said recombinant TCR or part thereof comprising: (i) a beta TCR (TCRβ) chain that comprises a beta variable domain (Vβ) and a beta constant domain (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the sequence nucleic acid comprising

(a) a transgene sequence encoding the TCRα and the Vβ domain, and (b) and an open reading frame of the endogenous TRBC locus or a partial sequence thereof, wherein the open reading frame encodes at least part the recombinant TCR Cβ domain. In some of any such modalities, a part of Cβ is encoded by the open reading frame of the endogenous TRBC locus or a partial sequence thereof, and another part of Cβ is encoded by the transgene sequence, in which said other part of Cβ is smaller than the natural size of a native Cβ; and the other part of Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, in some cases a substitution, deletion, or insertion of one or more amino acids compared to a region Native Cβ and / or a native Cα region, in some cases said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0024] In some of these modalities, the transgene sequence is or has been integrated by means of homology-directed repair (HDR). In some embodiments, the TRBC locus is a TRBC1 locus and / or a TRBC2 locus. In some embodiments, the modified TRBC locus contains a structure fusion of (i) a transgene sequence and (ii) and an open reading frame or a partial sequence thereof of the endogenous TRBC locus. In some modalities, the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the endogenous TRBC locus. In some embodiments, the transgene sequence does not contain a sequence that encodes a 3 'untranslated region (RTU 3') or an intron. In some embodiments, the open reading frame or a partial sequence thereof contains a 3 'RTU from the endogenous TRBC locus.

[0025] In some modalities, a part of the Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence of it, and another part of the Cβ is encoded by the transgene sequence, in which said other part of Cβ is smaller than the natural size of a native Cβ.

[0026] In some modalities, the open reading structure or the partial sequence thereof contains at least one intron and at least one exon from the endogenous TRBC locus. In some embodiments, the transgene sequence is in structure with one or more exons from the open reading frame or partial sequence from the endogenous TRBC locus. In some embodiments, the transgene sequence is or has been integrated downstream of the majority of 5 'nucleotide of exon 1 and upstream of the majority of 3' nucleotide of exon 1 of the open reading frame of the endogenous TRBC locus.

[0027] In some embodiments, said at least a part of Cβ is encoded by at least exons 2 to 4 of the open reading structure of the endogenous TRBC locus. In some embodiments, said at least a part of Cβ is encoded by at least a part of exon 1 and exons 2 to 4 of the open reading frame of the endogenous TRBC locus. In some embodiments, said at least a part of Cβ is encoded by less than the total length of exon 1 of the open reading frame of the endogenous TRBC locus.

[0028] In some of any such modalities, the other part of Cβ is encoded by a sequence of nucleotides that comprises less than four exons, less than three exons, less than two exons, one exon, or less than than a full exon of the open reading frame of a TRBC locus. In some of any such modalities, the other part of Cβ is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs in length. In some of any such modalities, the other part of the Cβ is encoded by an exon 1 part of a TRBC locus, where the exon 1 part is smaller than the natural exon 1 size of the open reading structure of the TRBC locus .

[0029] In some embodiments, the encoded TCRβ chain is able to dimerize with a TCRα chain.

[0030] In some embodiments, the encoded Cβ contains the selected sequence from any of SEQ ID NO: 16, 17, 20, 21, and 25, or a sequence that displays at least 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NO: 16 , 17, 20, 21, and 25, or a partial sequence of them.

[0031] In some embodiments, the encoded Cβ comprises the selected sequence from any of SEQ ID NO: 20, 21, 25 and 253-258 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NO: 20, 21, 25 and 253-258, or a partial sequence thereof.

[0032] In some embodiments, the other part of Cβ and / or said at least part of Cβ is encoded by a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons of the same or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof, or a partial sequence the same; or a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof or a sequence that exhibits at least

85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons of the same, or a partial sequence thereof. In some modalities, the other part of Cα is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs of lenght.

[0033] In some embodiments, the other part of Cα is encoded by a nucleotide sequence that contains less than four exons, less than three exons, less than two exons, one exon, or less than a complete exon of open reading structure of a TRBC locus. In some embodiments, the other part of Cα is encoded by an exon 1 part of a TRBC locus, where the exon 1 part is smaller than the natural exon 1 size of the open reading structure of the TRBC locus or the exon part 1 contains a 5 'portion of exon 1.

[0034] In some embodiments, the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) contains one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids in compared to a native Cβ region and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0035] In some of any modalities, the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. In some of any modalities, a coded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO:

20. In some of any modalities, the encoded Cβ comprises the selected sequence of any one of SEQ ID NOS: 253 and 256-258, or a partial sequence thereof.

[0036] In some embodiments, the modified T cell also contains a genetic disruption at a TRAC locus.

[0037] In some modalities, the transgene sequence contains one or more multicistronic elements. In some of these modalities, multicistronic elements are positioned between the nucleic acid sequence that encodes TCRα or a part of it and the nucleic acid sequence that encodes TCRβ or a part of it. In some embodiments, one or more multicistronic elements are upstream of the nucleic acid sequence that encodes the TCR or a part of the TCR or the nucleic acid molecule that encodes a TCR. In some embodiments, the multicistronic element is or contains a ribosome jump sequence, optionally T2A, P2A, E2A, or F2A.

[0038] In some modalities, the transgene sequence contains one or more heterologous control or regulatory elements. In some embodiments, the transgene sequence contains one or more heterologous or regulatory control elements operably linked to control TCR expression when expressed from a cell introduced with the genetically modified T cell. In some modalities, one or more heterologous control or regulatory elements contain a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence and / or a splice donor sequence.

[0039] In some modalities, the heterologous regulatory element

go or control contains a heterologous promoter, such as a heterologous promoter selected from a constitutive promoter, an inducible promoter, a repressible promoter, and / or a tissue-specific promoter. In some embodiments, the heterologous promoter is or contains a factor 1 alpha (EF1α) human elongation promoter or an MND promoter or a variant thereof.

[0040] In some modalities, the Cα and / or Cβ of the recombinant TCR contains one or more non-native cysteines.

[0041] In any of the modalities provided, the recombinant TCR may be able to bind to an antigen that is associated with, specific to, and / or expressed on a cell or tissue that is associated with a disease, disorder, or condition, such as an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer.

[0042] In some embodiments, the antigen is a tumor antigen or a pathogenic antigen, such as a bacterial antigen or viral antigen. In some modalities, where the antigen is a viral antigen, the viral antigen can optionally be from hepatitis A virus, hepatitis B, hepatitis C (HCV), human papilloma virus (HPV), hepatitis viral infections, Epstein virus -Barr (EBV), human herpesvirus virus 8 (HHV-8), human T cell leukemia virus 1 (HTLV-1), human T cell leukemia virus 2 (HTLV-2), or a cytomegalovirus (CMV).

[0043] In some embodiments, the viral antigen is an antigen from an HPV selected from among HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35, such as an HPV-16 antigen that is a HPV-16 E6 or HPV-16 E7 antigen. In some modalities, the viral antigen is an EBV antigen selected from the nuclear antigen Epstein-Barr (EBNA) -1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), proteins of latent membrane LMP-1, LMP-2A and

LMP-2B, EBV-EA, EBV-MA and EBV-VCA. In some embodiments, the viral antigen is an HTLV antigen that is TAX. In some modalities, the viral antigen is an HBV antigen that is a hepatitis B core antigen or a hepatitis B envelope antigen.

[0044] In some modalities, antigen is a tumor antigen. In some modalities, the antigen is selected from antigen associated with glioma, human chorionic gonadotropin β, alpha-fetoprotein (AFP), lectin-reactive AFP, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase , RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, Melanin-A / MART-1, WT-1, S-100, MBP, CD63, MUC1 (for example, MUC1- 8), p53, Ras, cyclin B1, HER-2 / neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE- A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE-1, GAGE- 2, p5, tyrosinase (for example, tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, p78 IFN inducible, melanotransferrin (p97), Uroplaquin II, prostate specific antigen (PSA), calic human kingdom (huK2), prostate-specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRa, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33 / IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin development factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin.

[0045] In any of the provided modalities, the T cell can be a primary T cell derived from an individual, optionally in which the individual is a human. In some embodiments, the T cell is a CD8 + T cell or subtypes thereof or a CD4 + T cell or subtypes thereof. In some embodiments, the T cell is derived from a multipotent or pluripotent cell, which is optionally an iPSC.

[0046] Compositions containing a plurality of genetically modified T cells, as well as a plurality of any of the modified cells provided in this document are also provided in this document. In some embodiments, compositions are provided that contain a plurality of genetically modified T cells comprising a modified T cell receptor alpha (TRAC) constant locus, the modified TRAC locus comprising a nucleic acid encoding a recombinant TCR or part thereof. therefrom, said recombinant TCR or part thereof comprising: (i) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), and (ii) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoding TCRβ and the Vα domain, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, where the open reading frame encodes at least part of the Cα domain of the recombinant TCR, and the recombinant TCR is capable of binding to an antigen that is associated with, speci co to, and / or expressed in, a cell or tissue that is associated with a disease, disorder, or condition. In some of any such modalities, at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells modified in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a benign constant region gene

T cell receptor (TRBC); at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express detectable levels of a gene product from an endogenous TRAC or TRBC gene. In some of any such modalities, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant TCR and / or exhibit binding to the antigen.

[0047] Compositions containing a plurality of genetically modified T cells comprising a modified alpha T cell receptor (TRAC) constant locus, said modified TRAC locus comprising an acid, are also provided in this document. nucleic encoding a recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) an alpha TCR chain (TCRα) comprising a variable alpha domain (Vα) and a constant alpha domain (Cα), and (ii) a beta TCR (TCRβ) chain comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoding TCRβ and the domain Vα, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, where the open reading frame encodes at least part of the Cα domain of the recombinant TCR. In some of any such modalities, a part of Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, where said other part of Cα is smaller than the natural size of a native Cα. In some of any such modalities, the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications compared to a native Cα region and / or a native Cβ region, referred to one or more modifications

Cations introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0048] Compositions containing a plurality of genetically modified T cells comprising a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus which comprises a nucleic acid encoding a recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a constant domain beta (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the nucleic acid sequence comprises (a) a transgene sequence encoding the TCRα and the Vβ domain, and (b) and an open reading frame of the endogenous TRBC site or a partial sequence thereof, where the open reading frame encodes at least a part of the Cβ domain recombinant TCR, and said recombinant TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder or condition. In some of any such modalities, at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells modified in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a T cell receptor beta constant region (TRBC) gene. In some of any such modalities, at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express detectable levels of an endogenous TRAC or TRBC gene product. In some of any such modalities,

at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant TCR and / or exhibit antigen binding.

[0049] Compositions containing a plurality of genetically modified T cells comprising a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus which comprises a nucleic acid encoding a recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a constant domain beta (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the nucleic acid sequence comprises (a) a transgene sequence encoding the TCRα and the Vβ domain, and (b) and an open reading frame of the endogenous TRBC site or a partial sequence thereof, where the open reading frame encodes at least a part of the Cβ domain recombinant TCR. In some of any such modalities, a part of the Cβ is encoded by the open reading frame of the endogenous TRBC locus or a partial sequence thereof, and another part of the Cβ is encoded by the transgene sequence, in which the said other part of Cβ is smaller than the natural size of a native Cβ. In some of any such modalities, the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids in comparison with a native Cβ region and / or a native Cα region. In some of these modalities, one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0050] In some of any modalities, the recombinant TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder or condition.

[0051] In some embodiments, provided in this document is a composition that contains a plurality of any of the genetically modified T cells provided in this document. In any of the embodiments, the composition contains CD4 + and / or CD8 + T cells. In some embodiments, the composition contains CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is about 1: 3 to 3: 1, optionally 1: 1.

[0052] In some modalities, the composition contains at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition that contain a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a constant T cell receptor beige gene ( TRBC); and / or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells modified in the composition do not express detectable levels of a gene product of an endogenous TRAC or TRBC gene. In some modalities, at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express recombinant TCR and / or exhibit antigen binding (for example, exhibit antigen binding).

[0053] In some embodiments, polynucleotides are provided, such as polynucleotides for use in modified cells. In some embodiments, polynucleotides are provided herein that contain (a) a nucleic acid sequence that encodes a part of a recombinant T cell receptor (TCR), said nucleic acid sequence that encodes (i) a T cell beta receptor chain (TCRβ) containing a beta variable domain (Vβ) and a beta constant domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is less than a full-length native TCRα chain, and (b) one or more homology arms attached to the nucleic acid sequence, wherein said one or more homology arms contain a sequence homologous to one or more regions of an open reading frame of a TRAC locus. In some modalities, the polynucleotide is comprised of a viral vector.

[0054] Provided herein are polynucleotides containing (a) a nucleic acid sequence that encodes a part of a recombinant T cell receptor (TCR), said nucleic acid sequence that encodes (i) a beta chain T cell receptor (TCRβ) comprising a variable beta domain (Vβ) and a constant beta domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is smaller than a natural size of a TCRα chain, and (b) one or more homology arms attached to the nucleic acid sequence, wherein said one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus; where, when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide: a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence of the same, and another part of Cα is encoded by the transgene sequence, in which said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, said one or more modifications included they introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. In some respects, and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more non-native cysteines.

[0055] Provided herein are polynucleotides containing (a) a nucleic acid sequence that encodes a part of a recombinant T cell receptor (TCR), said nucleic acid sequence that encodes (i) a beta chain T cell receptor (TCRβ) comprising a variable beta domain (Vβ) and a constant beta domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is smaller than a natural size of a TCRα chain, and (b) one or more homology arms attached to the nucleic acid sequence, wherein said one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus; wherein the transgene sequence comprises one or more heterologous or regulatory control elements comprising a heterologous promoter, operably linked to control TCR expression when expressed from a cell introduced with the genetically modified T cell.

[0056] In some embodiments, the TCRα chain contains a constant alpha region (Cα), in which at least part of said Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide.

[0057] In some embodiments, in which the nucleic acid sequence of (a) and the aforementioned one or more of the homology arms together contain a nucleotide sequence that encodes the

Cα that is smaller than the natural size of a native Cα, in which at least part of the Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof it is expressed from a cell introduced with the polynucleotide.

[0058] In some embodiments, the nucleic acid sequence that encodes the TCRβ chain is upstream of the nucleic acid sequence that encodes a part of the TCRα chain. In some modalities, the nucleic acid sequence of (a) does not contain an intron. In some embodiments, the nucleic acid sequence of (a) does not contain a sequence that encodes a 3 'untranslated region (3' UTR). In some embodiments, the nucleic acid sequence of (a) is a sequence that is exogenous or heterologous to an open reading frame of an endogenous genomic TRAC locus of a T cell, optionally a human T cell. In some embodiments, the nucleic acid sequence of (a) is in structure with one or more exons or a partial sequence thereof of the open reading frame of the TRAC locus contained in said one or more homology arms (s). In some embodiments, one or more exons or a partial sequence thereof from the open reading frame contains a sequence within exon 1 of the open reading frame of the TRAC location.

[0059] In some modalities, the TCRα chain is capable of dimming with a TCRβ chain, when produced from a cell introduced with the polynucleotide. In some embodiments, the TCRα chain contains a variable alpha domain (Vα).

[0060] In some embodiments, a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the nucleic acid sequence of (a), in that said other part of

Cα is smaller than the natural size of a native Cα. In some of any modalities, the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon from the endogenous TRAC locus. In some embodiments, the other part of Cα is encoded by a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof or a sequence that exhibits at least 85% , 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a sequence nucleotides starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof, or a partial sequence thereof. In some modalities, Cα is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs in length.

[0061] In some embodiments, the other part of Cα is encoded by a nucleotide sequence that contains less than four exons, less than three exons, less than two exons, one exon, or less than a complete exon of open reading structure of the TRAC locus. In some embodiments, the other part of Cα is encoded by an exon 1 part of the TRAC locus, where the exon 1 part is smaller than the natural exon 1 size of the open reading structure of the TRAC locus, such as as, in which the exon part 1 contains a 5 'portion of exon 1.

[0062] In some of any such modalities, the encoded Cα comprises the selected sequence from any of SEQ ID NOS: 19, 24 and 243 to 252, or a sequence that exhibits at least 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS: 19 , 24 and 243 to 252, or a partial sequence thereof, when produced from a cell introduced with the polynucleotide. In some of any such modalities, said at least a part of Cα is encoded by a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of same or a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof, or a partial sequence thereof.

[0063] In some embodiments, the other part of Cα contains a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91% , 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 142, or a partial sequence thereof.

[0064] In some embodiments, the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) contains one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids in compared to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0065] In some of any modalities, the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. In some of any modalities, a coded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds

from position 57 with numbering as mentioned in SEQ ID NO: 20, when produced from a cell introduced with the polynucleotide.

[0066] In some of any modalities, the encoded Cα comprises the sequence selected from any of SEQ ID NOS: 248-252, or a partial sequence thereof, when produced from a cell introduced with the polynucleotide. In some of any modalities, the part of the TCRα chain comprises a variable alpha domain (Vα).

[0067] In some embodiments, said one or more homology arms contains a 5 'homology arm and / or a 3' homology arm. In some embodiments, the 5 'homology arm and 3' homology arm contain nucleic acid sequences homologous to the nucleic acid sequences surrounding a target site, where the target site is within the TRAC locus, such as within of exon 1 of the TRAC locus.

[0068] In some embodiments, the 5 'homology arm contains: a) a sequence that contains at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides contiguous to a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more than sequence identity with the sequence mentioned in SEQ ID NO: 124; b) a sequence containing or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence mentioned in SEQ ID NO: 124; or c) the sequence mentioned in SEQ ID NO: 124.

[0069] In some embodiments, the 3 'homology arm contains: a) a sequence containing at or at least at or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides contiguous to a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%,

90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the sequence mentioned in SEQ ID NO: 125; b) a sequence containing or at least at or at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides from the sequence mentioned in SEQ ID NO: 125; or c) the sequence mentioned in SEQ ID NO: 125.

[0070] In some embodiments, provided in this document is a polynucleotide, containing: (a) a nucleic acid sequence that encodes a part of a recombinant T cell receptor (TCR), said nucleic acid sequence which encodes (i) a T cell receptor alpha chain (TCRα) containing a variable alpha domain (Vα) and a constant alpha domain (Cα); and (ii) a part of a T-cell receptor (TCRβ) beta chain, where the part of the TCRβ chain is less than a full-size native TCRβ chain, and (b) one or more homology arms attached to the nucleic acid sequence, wherein said one or more homology arms contain a sequence homologous to one or more regions of an open reading frame of a TRBC locus. In some of these modalities, the TCRβ chain contains a beta constant (Cβ), in which at least part of that Cβ is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence of the same when the TCR or fragment binding to it is expressed from a cell introduced with the polynucleotide. In some embodiments, the nucleic acid sequence of (a) and the aforementioned one or more homology arms together contain a nucleotide sequence that encodes Cβ which is smaller than the natural size of a Cβ native, in which at least part of the Cβ is encoded by the open reading frame of the endogenous TRBC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from an intro cell

produced with the polynucleotide.

[0071] In some embodiments, the TRBC locus is one or more of TRBC1 or TRBC2.

[0072] In some embodiments, the nucleic acid sequence encoding the TCRα chain is upstream of the nucleic acid sequence encoding the part of the TCRβ chain. In some modalities, the nucleic acid sequence of (a) does not contain an intron. In some embodiments, the nucleic acid sequence of (a) does not contain a sequence that encodes a 3 'untranslated region (3' UTR). In some embodiments, the nucleic acid sequence of (a) is a sequence that is exogenous or heterologous to an open reading frame of an endogenous genomic TRBC locus of a T cell, optionally a human T cell.

[0073] In some embodiments, the nucleic acid sequence of (a) is in structure with one or more exons or a partial sequence thereof from the open reading structure of the TRAC locus contained in said one or more homology arms ( s). In some embodiments, one or more exons or a partial sequence of the same open reading frame is or contains a sequence within exon 1 of the open reading frame of the TRBC locus.

[0074] In some modalities, the TCRβ chain is capable of dimming with a TCRα chain, when produced from a cell introduced with the polynucleotide. In some of any modalities, the encoded Cβ comprises the sequence selected from any of SEQ ID NO: 20, 21, 25 and 253-258 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NO: 20, 21, 25 and 253-258, or a partial sequence thereof, when produced from a cell introduced with the polynucleotide.

[0075] In some embodiments, the part of the TCRβ chain contains a variable beta domain (Vβ). In some embodiments, a part of Cβ is encoded by the open reading frame of the ingenious TRBC locus or a partial sequence thereof, and another part of Cβ is encoded by the nucleic acid sequence of (a), in which I refer to it - the other part of Cβ is smaller than the natural size of a native Cβ. In some of any embodiments, the open reading frame or partial sequence thereof comprises at least one intron and at least one exon from the endogenous TRBC locus. In some of these modalities, the other part of Cβ is encoded by: a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons of the same or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity sequence with a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof, or a partial sequence thereof; or a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof, or a partial sequence thereof.

[0076] In some of these modalities, the other part of Cβ is codified by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs in length.

[0077] In some embodiments, the other part of Cβ is encoded by a nucleotide sequence that encodes less than four exons, less than three exons, less than two exons, one exon, or less than a complete exon of open reading structure of the TRBC locus. In some embodiments, the other part of the Cβ is encoded by an exon 1 part of a TRBC locus, where the exon 1 part is smaller than the natural exon 1 size of the open reading structure of the TRBC locus. In some embodiments, the other part of the TCRα constant domain is encoded by an exon 1 part of the TRAC locus, where exon 1 part contains a 5 'exon portion

1.

[0078] In some embodiments, the other part of Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) contains one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids in compared to a native Cβ region and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[0079] In some of any modalities, the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. In some of any modalities, a coded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO:

20. In some of any modalities, the encoded Cβ comprises the selected sequence of any one of SEQ ID NOS: 253 and 256-258, or a partial sequence thereof. In some of any modalities, the part of the TCRβ chain comprises a

variable beta domain (Vβ).

[0080] In some embodiments, said one or more homology arms contain a 5 'homology arm and / or a 3' homology arm. In some embodiments, the 5 'homology arm and 3' homology arm contain nucleic acid sequences homologous to the nucleic acid sequences surrounding a target site, where the target site is within the open reading frame of the TRBC locus. . In some modalities, the target site is within exon 1 of the open reading structure of the TRBC locus.

[0081] In some embodiments, the 5 'homology arm and 3' homology arm independently are at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less than or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides. In some of these modalities, the 5 'homology arm and 3' homology arm regardless of or from about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides or 750 to 1000 nucleotides in length. In some embodiments, the homology arm 5 'and homology arm 3' independently are, are about, or are less than about 600 nucleotides in length.

[0082] In some of any embodiments, the nucleic acid sequence of (a) is a sequence that is exogenous or heterologous to an open reading frame of an endogenous genomic TRAC locus of a T cell, optionally a human T cell . In some of any embodiments, the 5 'homology arm and the 3' homology arm independently are at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less than or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides. In some of any embodiments, the 5 'homology arm and the 3' homology arm independently are between 50 or about and 100 or about, nucleotides in length, 100 or about, and 250 or about, nucleotides of length, 250 or about, and 500 or about, nucleotides in length, 500 or about, and 750 or about, nucleotides in length, 750 or about, and 1,000 or about, nucleotides in length, 1,000 or about of, and 2,000 or about, nucleotides in length. In some of any embodiments, the 5 'homology arm and the 3' homology arm independently are either about 200, 300, 400, 500, 600, 700 or 800 nucleotides in length, or any value between any of the above. In some of any embodiments, the 5 'homology arm and the 3' homology arm are independently greater than or about 300 nucleotides in length, optionally where the 5 'homology arm and the homology arm 3 'independently are either about 400, 500 or 600 nucleotides in length or any value between any of the foregoing. In some of any such embodiments, the 5 'homology arm and the 3' homology arm are independently greater than or about 300 nucleotides in length.

[0083] In some embodiments, the nucleic acid sequence of (a) contains one or more multicistronic elements. In some embodiments, the multicistronic elements are positioned between the nucleic acid sequence that encodes TCRα or a part of it and the nucleic acid sequence that encodes TCRβ or a part of it. In some embodiments, one or more multicistronic elements are upstream of the nucleic acid sequence that encodes the TCR or a part of the TCR or the nucleic acid molecule that encodes a TCR. In some embodiments, the multicistronic element is or contains a ribosome jump sequence, optionally T2A, P2A, E2A, or F2A.

[0084] In some embodiments, the polynucleotide provided also contains one or more heterologous control or regulatory elements. In some embodiments, the nucleic acid sequence of (a) contains one or more heterologous or regulatory control elements operably linked to control TCR expression when expressed from a cell introduced with the polynucleotide, such as a promoter, a enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence and / or a splice donor sequence. In some modalities, the heterologous or control regulatory element contains a heterologous promoter, such as, a heterologous promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and / or a tissue specific promoter. In some modalities, the heterologous promoter is or contains a factor 1 alpha (EF1α) human length promoter or an MND promoter or a variant thereof.

[0085] Any of the supplied polynucleotides may be contained in a viral vector. In some embodiments, the viral vector is an AAV vector, such as an AAV vector selected from the vector AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8. In some modalities, the AAV vector is an AAV2 or AAV6 vector.

[0086] In some embodiments, the viral vector is either a retroviral vector or a lentiviral vector.

[0087] In some embodiments, the polynucleotide is a linear polynucleotide. In some embodiments, the linear polynucleotide is a double-stranded polynucleotide or a single-stranded polynucleotide.

[0088] In some embodiments, the polynucleotide contains the structure: [5 'homology arm] - [nucleic acid sequence of (a)] - [3' homology arm]. In some embodiments, the polynucleotide contains the structure: [5 'homology arm] - [multicistronic element] - [nucleic acid sequence from (a)] - [3' homology arm]. In some embodiments, the polynucleotide contains the structure: [5 'homology arm] - [promoter] - [nucleic acid sequence of (a)] - [3' homology arm].

[0089] In some embodiments, the recombinant TCR encoded by the polynucleotide is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition, such as an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor or a cancer.

[0090] In some embodiments, the antigen is a tumor antigen or a pathogenic antigen. In some of these modalities, the pathogenic antigen is a bacterial or viral antigen. In some embodiments, the antigen is a viral antigen, optionally a viral antigen from hepatitis A virus, hepatitis B, hepatitis C (HCV), human papilloma virus (HPV), hepatitis viral infections, Epstein-Barr virus (EBV) ), human herpes virus 8 (HHV-8), human T cell leukemia virus 1 (HTLV-1), human T cell leukemia virus 2 (HTLV-2) or a cytomegalovirus (CMV). In some modalities, the antigen is an antigen from an HPV selected from HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35, such as an HPV-16 antigen that is an antigen from HPV-16 E6 or HPV-16 E7.

[0091] In some modalities, the viral antigen is an EBV antigen selected from the nuclear antigen Epstein-Barr (EBNA) -1, EB-NA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA - LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA. In some embodiments, the viral antigen is an HTLV antigen that is TAX.

[0092] In some embodiments, the viral antigen is an HBV antigen that is a hepatitis B core antigen or a hepatitis B envelope antigen.

[0093] In some embodiments, the antigen is a tumor antigen, such as, an antigen is selected from an antigen associated with the glioma, human chorionic gonadotropin β, alpha fetoprotein (AFP), AFP reactive to lectin, thyroglobulin, RAGE-1, MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, Melanin-A / MART-1, WT-1, S-100, MBP , CD63, MUC1 (e.g., MUC1-8), p53, Ras, cyclin B1, HER-2 / neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE- C1, BAGE, GAGE-1, GAGE-2, p5, tyrosinase (for example, tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY- ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, p78 IFN inducible, melanotransferrin (p97), Uroplaquin II, prostate specific antigen (PSA), human kallikrein (huK2), prostate specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A- PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRa, CD24, CD44,

CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33 / IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin development factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin.

[0094] In some of any embodiments, the polynucleotide provided is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4,250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000 , 7500, 8000, 9000 or 10,000 nucleotides in length, or any value between any of the above. In some of any modalities, the polynucleotide is between or about 2,500, and of or about 5,000 nucleotides, of or about 3,500 and of or about 4,500 nucleotides, or of or about 3,750 nucleotides and of or about in

4,250 nucleotides in length.

[0095] In some embodiments, provided in this document is a method of producing a genetically modified T cell that contains a modified TRAC locus, which contains introduction of any of the nucleotides provided into a T cell that contains a genetic disruption in a TRAC locus.

[0096] In some embodiments, provided in this document is a method of producing a genetically modified T cell that contains a modified alpha T cell receptor (TRAC) constant locus, the method including: (a ) introducing, in a T cell, one or more agents capable of inducing a genetic disruption at a target site within an endogenous TRAC locus of the T cell; and (b) introducing into the T cell a polyucleotide containing a transgene encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), where the part is smaller than that a native TCRα chain of natural size, and in which the transgee is directed to integration within the endogenous TRAC locus by means of homology-directed repair (HDR), thereby producing

making a genetically modified cell that contains a modified TRAC locus.

[0097] In some modalities, the introduction of the standard polynucleotide is carried out after the introduction of said one or more agents capable of inducing a genetic rupture.

[0098] Methods are also provided in this document, such as methods of producing a genetically modified T cell. In some embodiments, methods are provided to produce a genetically modified T cell that comprises a modified alpha T cell receptor (TRAC) constant locus, the method comprising: (a) introducing into a T cell, one or more agents capable of induce a genetic disruption at a target site within an endogenous T cell TRAC locus; and (b) introducing into the T cell a polyucleotide comprising a transgene sequence encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), wherein the part is smaller than a native, full-size TCRα chain; and the transgeon is directed towards integration within the endogenous TRAC locus by means of repair directed to homology (HDR); thereby producing a genetically modified cell that comprises a modified TRAC locus. In some of these modalities, after targeted integration of the transgene: a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence of it, and another part of Cα is encoded by the transgene sequence, in that said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, said one or more further modifications introduce one or more cysteine residues that are capable of forming

marking one or more non-native disulfide bridges between the alpha chain and the beta chain. In some embodiments, the Cα and / or Cβ of the recombinant TCR comprises one or more non-native cysteines.

[0099] Also provided are methods of producing a genetically modified T cell that comprises a modified alpha T cell receptor (TRAC) constant locus, the method of introducing into a T cell a polynucleotide that comprises a transgene sequence that encodes a T cell receptor beta chain (TCRβ) and part of a T cell receptor alpha chain (TCRα), said cell having a genetic disruption within the endogenous T cell TRAC locus; and the transgene is directed towards integration within the endogenous TRAC locus through homology-directed repair (HDR); thereby producing a genetically modified cell that comprises a modified TRAC locus, in which, after targeted integration of the transgene: a part of the Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, where said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the corresponding TCR Cα and / or Cβ comprises one or more more non-native cysteines.

[00100] In some embodiments, provided in this document is a method of producing a genetically modified T cell that contains a modified alpha T cell receptor (TRAC) constant locus, the method that contains introducing, in a T cell, a polynucleotide that contains a transgene that encodes a nice T cell receptor chain (TCRβ) and part of an alpha T cell receptor chain (TCRα), said cell having a genetic disruption within the endogenous TRAC locus of the T cell, where the transgene is targeted for integration within the endogenous TRAC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that contains a modified TRAC locus. In some of these modalities, the genetic disruption was induced by one or more agents capable of inducing a genetic disruption of one or more target sites within the endogenous TRAC locus.

[00101] In some modalities of the methods provided, the polynucleotide is from any of the polynucleotides provided in this document.

[00102] In some embodiments of the methods provided, the modified TRAC locus contains a nucleic acid sequence that encodes a recombinant TCR or part thereof, wherein the nucleic acid sequence contains a structure fusion of (i) a sequence of transgene and (ii) and an open reading frame or a partial sequence thereof of the endogenous TRAC locus. In some modalities, the transgene sequence does not contain a sequence that encodes a 3 'untranslated region (RTU 3') or an intron. In some embodiments, the open reading frame or a partial sequence thereof contains a 3 'RTU from the endogenous TRAC locus. In some embodiments, the transgene sequence is structured with one or more exons from the open reading frame or partial sequence from the endogenous TRAC locus.

[00103] In some modalities, a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence of it, and another part of Cα is encoded by the transgene sequence, in which said other part of Cα is smaller than the natural size of a native Cα. In some modalities, the open reading frame or the partial sequence of the same contains at least one intron and at least one exon of the endogenous TRAC locus. In some modalities, the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the endogenous TRAC locus. In some embodiments, the transgene sequence is or has been integrated downstream of the majority of exon 5 'nucleotide 1 and upstream of the exon 1' 3 'nucleotide majority of the open reading frame of the endogenous TRAC locus. In some embodiments, said at least a part of Cα is encoded by at least exons 2 to 4 of the open reading frame of the endogenous TRAC locus.

[00104] In some modalities of the methods provided, the encoded Cα contains the selected sequence from any of SEQ ID NOS: 14, 15, 19, or 24, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS: 14, 15, 19, or 24, or a partial sequence of them. In some embodiments, the other part of Cα contains a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 142, or a partial sequence thereof.

[00105] In some modalities of the methods provided, the modified T cell also contains induction of a genetic disruption at a TRBC locus. In some embodiments, the modified T cell contains a genetic disruption at a TRBC1 locus and / or a TRBC2 locus.

[00106] In some embodiments, provided in this document is a method of producing a genetically modified T cell that contains a modified TRBC locus, which contains introduction of any of the polynucleotides provided herein into a T cell that contains a genetic disruption at a TRBC locus.

[00107] In some embodiments, provided in this document is a method of producing a genetically modified T cell containing a modified T cell receptor (TRBC) beta constant locus, the method including: (a) introducing, in a T cell, one or more agents capable of inducing a genetic disruption at a target site within an endogenous TRBC locus of the T cell; and (b) introducing into the T cell a polyucleotide containing a transgene encoding a T cell receptor alpha chain (TCRα) and a part of a T cell receptor beta chain (TCRβ), where the part is less than that a natural-sized native TCRβ chain, and in which the transgene is targeted for integration into an endogenous TRBC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that contains a modified TRBC locus.

[00108] In some embodiments, provided in this document is a method of producing a genetically modified T cell that contains a modified T cell receptor (TRBC) beta constant locus, the method that contains introducing into a T cell a polynucleotide that contains a transgene that encodes a T cell receptor alpha chain (TCRα) and a part of a T cell receptor beta chain (TCRβ), said cell having a genetic disruption within an endogenous TRBC locus of the T cell, in which the transgene is directed to integration within the endogenous TRBC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that contains a modified TRBC locus. In some of these modalities, genetic disruption was induced by one or more agents capable of inducing a genetic disruption of one or more target sites within the TRBC locus involved.

dogen. In some modalities, the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the endogenous TRBC locus. In some embodiments, the TRBC locus is a TRBC1 locus and / or a TRBC2 locus.

[00109] In some embodiments, the polynucleotide is any of the polynucleotides provided in this document.

[00110] In some embodiments of the method provided, the modified TRBC locus contains a nucleic acid sequence that encodes a recombinant TCR or part thereof, wherein the nucleic acid sequence contains a structure fusion of (i) a sequence of transgene and (ii) and an open reading frame or partial sequence thereof from the endogenous TRBC locus. In some modalities, the transgene sequence does not contain a sequence that encodes a 3 'untranslated region (RTU 3') or an intron. In some embodiments, the open reading frame or a partial sequence thereof contains a 3 'RTU from the endogenous TRBC locus. In some embodiments, a part of Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of Cβ is encoded by the transgene sequence, where said other part of Cβ is smaller than the natural size of a native Cβ.

[00111] In some modalities, the open reading structure or the partial sequence of the same contains at least one intron and at least one exon of the endogenous TRBC locus. In some embodiments, the transgene sequence is in structure with one or more exons from the open reading frame or partial sequence from the endogenous TRBC locus. In some embodiments, the transgene sequence is or has been integrated downstream of the majority of 5 'nucleotide of exon 1 and upstream of the majority of 3' nucleotide of exon 1 of the open reading frame of the endogenous TRBC locus.

[00112] In some modalities of the methods provided, said at least a part of Cβ is encoded by at least exons 2 to 4 of the open reading structure of the endogenous TRBC locus. In some embodiments, the encoded Cβ contains the sequence selected from any of SEQ ID NO: 16, 17, 20, 21, or 25, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 16, 17, 20, 21, or 25, or a partial sequence thereof.

[00113] In some embodiments of the methods provided, the modified T cell also contains induction of a genetic disruption at a TRAC locus.

[00114] In some embodiments of the methods provided, said one or more agents capable of inducing a genetic disruption contains a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the site target. In some embodiments, said one or more agents capable of introducing a genetic disruption contain (a) a fusion protein containing a protein targeting DNA and a nuclease or (b) an RNA-guided nuclease. In some of these modalities, the protein targeting DNA or RNA-guided nuclease contains a zinc-depleted protein (ZFP), a TAL protein, or a nuclease associated with the short palindromic nucleic acid regularly interspersed in a group (CRISPR) (Cas) specific to the target site. In some modalities, said one or more agents contains a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or a combination of CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes to the target site.

[00115] In some embodiments, each of the said one or more agents contains a guide RNA (gRNA) having a direction domain.

which is complementary to at least one target site. In some embodiments, said one or more agents are introduced as a ribonucleoprotein complex (RNP) that contains the gRNA and a Cas9 protein. In some modalities, an RNP is introduced by means of electroporation, particle gun, calcium phosphate transfection, compression or cellular tightening.

[00116] In some embodiments, said one or more agents are introduced as one or more polynucleotides that encode a gRNA and / or a Cas9 protein. In some modalities, the gRNA has a targeting domain that is complementary to a target site in a TRAC locus and contains a selected sequence from UCUCUCAGCUGGUACACGGC (SEQ ID NO: 28), UGGAUUUAGA- GUCUCUCAGC (SEQ ID NO: 29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO: 30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31), GCUGGUACACGGCAGGGUCA (SEQ ID NO: 32), CUCAGCUGGUA- CACGGC (SEQ ID NO: 33), SEAGGGAC (34) ID NO: 35), GUCAGAUUU- GUUGCUCC (SEQ ID NO: 36), UCAGCUGGUACACGGCA (SEQ ID NO: 37), GCAGACAGACUUGUCAC (SEQ ID NO: 38), GGUACACGG- CAGGGUCA (SEQ ID NO: 39), CUUCAAGAGAGAGAGAGAGAGAGAGAC NO: 40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO: 41), AAA- GUCAGAUUUGUUGCUCC (SEQ ID NO: 42), ACAAAACUGUGCUA- GACAUG (SEQ ID NO: 43), AAACUGUGCUAGACAUG (SEQ ID NO: 44), UGUGGAGACUA : 45), GGCUG- GGGAAGAAGGUGUCUUC (SEQ ID NO: 46), GCUGGGGA- AGAAGGUGUCUUC (SEQ ID NO: 47), GGGGAAGAAGGUGUCUUC (SEQ ID NO: 48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO: 49) : 50), GCAGACA- GACUUGUCACUGGAUU (SEQ ID NO: 51), GACAGACUUGUCA- CUGGAUU (SEQ ID NO: 52), GUGAAUAGGCAGACAGACUUGUCA

(SEQ ID NO: 53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO: 54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO: 55), GUCUCUCAG- CUGGUACACGG (SEQ ID NO: 56), GGUACACGGCAGGGUCA- GGGU SEQ ID NO: 58). In some embodiments, the gRNA has a targeting domain that contains the sequence GAGAAUCAAAAUCGGU-GAAU (SEQ ID NO: 31).

[00117] In some embodiments, the gRNA has a targeting domain that is complementary to a target site in one or both of a TRBC1 and a TRBC2 gene and contains a selected sequence from CACCCAGAUCGUCAGCGCCG (SEQ ID NO: 59), CAAACACAGCGACCUCGGGU (SEQ ID NO: 60), UGACGAGUGGACCCAGGAUA (SEQ ID NO: 61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO: 62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63), GAAAAACGUGUUCCCACCCG (SEQ ID NO: 64), SEAG ID NO: 64), , AGUCCAGUUCUACGGGCUCU (SEQ ID NO: 66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO: 67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO: 68), UCAAACACAGCGACCUCGGG (SEQ ID NO: 69), CGUAGACGGGAC (70) NO: 71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO: 72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO: 73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO: 74), CGGGUGGGAACACGUUUUCUC (SEQ ID NO: 76), SEAGU GAUCGUCAGCGCCGAGGCCU (SEQ ID NO: 77),

GGCUCAAACACAGCGACCUC (SEQ ID NO: 78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO: 79), AGGCUUCUACCCCGACCACG (SEQ ID NO: 80), CCGACCACGUGGAGCUGAGC (SEQ ID NO: 81), SEA ID: 82), UGGGAGGU : 83), AGAUCGUCAGCGCCGAGGCC (SEQ ID NO: 84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO: 85), UGAGGGCGGGCUGCUCCUUG (SEQ ID NO: 86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO: 88), AGCGGG (SEQ ID NO: 89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO: 90), UGGCUCAAACACAGCGACCU (SEQ ID NO: 91), ACUGGACUUGACAGCGGAAG (SEQ ID NO: 92), GACAGCGGAAGUGGUUGCGG (SEUC ID: 93) 94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO: 95), CUCGGCGCUGACGAUCU (SEQ ID NO: 96), CCUCGGCGCUGACGAUC (SEQ ID NO: 97), CCGAGAGCCCGUAGAAC (SEQ ID NO: 98), CCAGAUCGACAGAGGGAGA SEQ ID NO: 100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO: 103), GACAGGUUUGGCCCUAUC (SEQ ID NO: 104) GAGCAGCCGCCU (SEQ ID NO: 105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO: 106), GUGGAGCUGAGCUGGUGG (SEQ ID NO: 107),

GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO: 112), GCUGCUCCUUGAGGGGCU (SEQ ID NO : 113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 116). In some embodiments, the gRNA has a targeting domain that contains the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63).

[00118] In some of any embodiments, the T cell is an individual's primary T cell. In some of any modalities, the individual has or is suspected of having the disease, disorder or condition. In some of any modalities, the individual is, or is suspected of being healthy.

[00119] In some modalities of the method, the T cell is a CD8 + T cell, or a subtype thereof, or is a CD4 + T cell, or a subtype thereof. In some embodiments, the T cell is derived from a multipotent or pluripotent cell, which is optionally an iPSC. In some modalities of the method, the T cell is autologous to the individual, or allogeneic to the individual.

[00120] In some embodiments of the method, the polynucleotide and / or one or more polynucleotides encoding a gRNA and / or a Cas9 protein is contained in one or more vectors, which are optionally viral vectors, such as an AAV vector. In some embodiments, the AAV vector is selected from the vector AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8, such as an AAV2 or AAV6 vector. In some embodiments, the viral vector is either a retroviral vector or a lentiviral vector.

[00121] In some embodiments of the method, the polynucleotide is a linear polynucleotide, such as a double-stranded polynucleotide or a single-stranded polynucleotide.

[00122] In some modalities of the methods provided, the introduction of said one or more agents capable of inducing a genetic rupture and an introduction of the standard polynucleotide are carried out simultaneously or sequentially, in any order. In some embodiments, the introduction of the standard polynucleotide is carried out after the introduction of said one or more agents capable of introducing a genetic disruption. In some embodiments, the standard polynucleotide is introduced immediately after, or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of one or more agents capable of introducing a rupture genetics.

[00123] In some embodiments, provided in this document is a modified T cell or a plurality of modified T cells generated using any of the methods provided in this document.

[00124] In some embodiments, provided in this document is a composition, which contains a modified T cell or plurality of modified cells provided in this document. In some embodiments, the composition provided contains CD4 + and / or CD8 + T cells. In some embodiments, the composition contains CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is about 1: 3 to 3: 1, optionally 1: 1.

[00125] In some modalities of the compositions provided, at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition contain a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a T cell receptor constant beta region (TRBC) gene; and / or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express detectable levels of a gene product of an endogenous TRAC or TRBC gene.

[00126] In some modalities of the compositions provided, at least less than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90 % of the cells in the composition express the recombinant TCR and / or exhibit antigen binding (for example, exhibit antigen binding).

[00127] In some embodiments, provided in this document is a treatment method that contains administration of any of the modified cells, plurality of modified cells or compositions provided in this document to an individual.

[00128] In some embodiments, provided in this document is a use of any of the modified cells, plurality of modified cells or compositions provided in this document for the treatment of a disease or disorder.

[00129] In some embodiments, provided in this document is a use of any of the modified cells, plurality of modified cells or compositions provided in this document in the manufacture of a drug to treat a disease or disorder.

[00130] In some embodiments, provided in the present document is a modified cell, plurality of modified cells or composition of any of the modified cells, plurality of modified cells, or compositions provided herein for use in the treatment of a disease or disorder.

[00131] In some embodiments, provided in this document is an article of manufacture or a kit containing a polynucleotide

tide provided in this document, and one or more agents capable of inducing a genetic disruption at a target site within a TRAC site.

[00132] In some embodiments, provided in this document is an article of manufacture or a kit containing a polynucleotide that contains (a) a nucleic acid sequence that encodes a beta cell T-receptor (TCRβ) chain and a portion of an alpha T cell receptor (TCRα) chain, where the portion is smaller than a native, full-size TCRα chain and (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homology arms (s) contain a sequence homologous to one or more regions of an open reading frame of a TRAC locus, said open reading frame encoding a TCRα chain; and one or more agents capable of inducing a genetic disruption at a target site within a TRAC locus. In some of these modalities, the polynucleotide is a polynucleotide provided herein.

[00133] In some embodiments, provided in this document is an article of manufacture that contains a polynucleotide provided in this document, and one or more agents capable of inducing a genetic disruption at a target site within a TRBC locus.

[00134] In some embodiments, provided in this document is an article of manufacture that contains a polynucleotide that contains (a) a nucleic acid sequence that encodes an alpha T cell receptor (TCRα) chain and a part of a beta cell receptor (TCRβ) chain, wherein the portion is less than a full-length native TCRβ chain and (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homology arms contain a sequence homologous to one or more regions of an open reading frame of a locus

TRBC, said open reading structure that encodes a TCRβ chain; and one or more agents capable of inducing a genetic disruption at a target site within a TRAC locus. In some embodiments, the TRBC locus is TRBC1 and / or TRBC2. In some modalities, the polynucleotide is selected from the polynucleotides provided in this document.

[00135] In some embodiments, said one or more agents capable of inducing a genetic disruption contains a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site. In some embodiments, said one or more agents capable of introducing a genetic disruption contain (a) a fusion protein that contains a protein targeting DNA and a nuclease or (b) an RNA-guided nuclease. In some of these modalities, the protein targeting DNA or RNA-guided nuclease contains a zinc finger protein (ZFP), a TAL protein, or a nuclease associated with the short palindromic nucleic acid regularly interspersed in a group (CRISPR) (Cas) specific to the target site. In some embodiments, said one or more agents contain a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or a combination of CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes to the target site .

[00136] In some embodiments, each of the said one or more agents contains a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. In some embodiments, said one or more agents are introduced as a ribonucleoprotein complex (RNP) that contains the gRNA and a Cas9 protein. In some modalities, RNP is introduced by means of electroporation, particulate weapon, calcium phosphate transfection, compression or cellular tightening.

[00137] In some embodiments, said one or more agents are introduced as one or more polynucleotides encoding a gRNA and / or a Cas9 protein. In some embodiments, the gRNA has a targeting domain that is complementary to a target site on a TRAC locus and contains a selected sequence from UCUCUCAGCUGGUACACGGC (SEQ ID NO: 28), UGGAUUUAGAGUCUCUCAGC (SEQ ID NO: 29), ACACGGCAGGGUCAGGGUC ( SEQ ID NO: 30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31), GCUGGUACACGGCAGGGUCA (SEQ ID NO: 32), CUCAGCUGGUACACGGC (SEQ ID NO: 33), UGGUACACGGAGAGGUC (SEQ ID NO: 35) ), GUCAGAUUUGUUGCUCC (SEQ ID NO: 36), UCAGCUGGUACACGGCA (SEQ ID NO: 37), GCAGACAGACUUGUCAC (SEQ ID NO: 38), GGUACACGGCAGGGUCA (SEQ ID NO: 39), CUUCAAGAGCAACAGUGUGGUG (SEQ: 40) ID NO: 41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO: 42), ACAAAACUGUGCUAGACAUG (SEQ ID NO: 43), AAACUGUGCUAGACAUG (SEQ ID NO: 44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO: 45), GGCAGGGGGGGUA , GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO: 47), GGGGAAGAAGGUGUCUUC (SEQ ID NO: 48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO: 49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO: 50), GCAGACAGACU UUGUCACUGGAUU (SEQ ID NO: 51), GACAGACUUGUCACUGGAUU (SEQ ID NO: 52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO: 53),

GAAUAGGCAGACAGACUUGUCA (SEQ ID NO: 54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO: 55), GUCUCUCAGCUGGUACACGG (SEQ ID NO: 56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO: 58) and GUAC In some embodiments, the gRNA has a targeting domain that contains the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31).

[00138] In some embodiments, the gRNA has a targeting domain that is complementary to a target site in one or both of a TRBC1 and a TRBC2 gene and contains a selected sequence from CACCCAGAUCGUCAGCGCCG (SEQ ID NO: 59), CAAACACAGCGACCUCGGGU (SEQ ID NO: 60), UGACGAGUGGACCCAGGAUA (SEQ ID NO: 61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO: 62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63), GAAAAACGUGUUCCCACCCG (SEQ ID NO: 64) 65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO: 66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO: 67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO: 68), UCAAACACAGCGACCUCGGG (SEQ ID NO: 69), SEQ ID NO: 69), CGQGA SEQ ID NO: 71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO: 72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO: 73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO: 74), CGGGUGGGAACACGUUUUC (SEQ ID NO: 75) ), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO: 77),

GGCUCAAACACAGCGACCUC (SEQ ID NO: 78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO: 79), AGGCUUCUACCCCGACCACG (SEQ ID NO: 80), CCGACCACGUGGAGCUGAGC (SEQ ID NO: 81), SEA ID: 82), UGGGAGGU : 83), AGAUCGUCAGCGCCGAGGCC (SEQ ID NO: 84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO: 85), UGAGGGCGGGCUGCUCCUUG (SEQ ID NO: 86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO: 88), AGCGGG (SEQ ID NO: 89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO: 90), UGGCUCAAACACAGCGACCU (SEQ ID NO: 91), ACUGGACUUGACAGCGGAAG (SEQ ID NO: 92), GACAGCGGAAGUGGUUGCGG (SEUC ID: 93) 94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO: 95), CUCGGCGCUGACGAUCU (SEQ ID NO: 96), CCUCGGCGCUGACGAUC (SEQ ID NO: 97), CCGAGAGCCCGUAGAAC (SEQ ID NO: 98), CCAGAUCGACAGAGGGAGA SEQ ID NO: 100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO: 103), GACAGGUUUGGCCCUAUC (SEQ ID NO: 104) GAGCAGCCGCCU (SEQ ID NO: 105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO: 106), GUGGAGCUGAGCUGGUGG (SEQ ID NO: 107),

GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO: 112), GCUGCUCCUUGAGGGGCU (SEQ ID NO : 113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 116). In some embodiments, the gRNA has a targeting domain that contains the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63).

[00139] In some modalities, provided in this document is a kit that contains an article of manufacture provided in this document and instructions for use. In some of such embodiments, the instructions specify that said one or more agents and the polynucleotide are introduced into the cell. In some embodiments, the instructions specify that said one or more agents and the polynucleotide are introduced simultaneously or sequentially, in any order. In some embodiments, the instructions specify that the introduction of the polynucleotide is carried out after the introduction of said one or more agents. In some of these modalities, the instructions specify that the polynucleotide is introduced immediately after, or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 8 minutes, 9 mi - nutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of one or more agents capable of introducing a genetic disruption.

[00140] In some of any such modalities, the polynucleotide is introduced in or about 2 hours after the introduction of the references

one or more agents.

[00141] In some of any modalities, the method is performed on a plurality of T cells. In some of any modalities, the plurality of T cells comprise CD4 + T cells, CD8 + T cells or CD4 + T cells and CD8 +. In some of any embodiments, the plurality of T cells comprise CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is about 1: 3 to about 3: 1, optionally about 1: 2 to about 2: 1, optionally about or about 1: 1.

[00142] In some of any modalities, before the introduction of said one or more agents, the method comprises incubating the cells in vitro with a stimulating agent under conditions to stimulate or activate said one or more T cells. in any embodiment, the stimulatory agents comprise anti-CD3 and / or anti-CD28 antibodies, optionally anti-CD3 / anti-CD28 beads, optionally wherein the bead to cell ratio is, or is about 1: 1 . In some of any embodiments, the methods include removing stimulatory agents from one or more immune cells prior to introduction with one or more agents.

[00143] In some of any modalities, the method also comprises incubating the cells before, during or subsequent to the introduction of one or more agents and / or the introduction of the polynucleotide pattern with one or more recombinant cytokines, optionally wherein one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15.

[00144] In some of any embodiments, one or more recombinant cytokines are added at a selected concentration from an IL-2 concentration of or about 10 U / mL to or about 200 U / mL, optionally from or about 50 IU / ml to or about 100 U / ml; IL-7 at a concentration of 0.5 ng / mL to 50 ng / mL, optionally from or about 5 ng / mL to or about 10 ng / mL and / or IL-15 at a concentration of 0.1 ng / ml to 20 ng / ml, optionally from or about 0.5 ng / ml to or about 5 ng / ml.

[00145] In some of any modalities, incubation is performed subsequent to the introduction of one or more agents and the introduction of the polynucleotide pattern for up to or approximately 24 hours, 36 hours, 48 hours, 3, 4, 5 , 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, optionally up to or about 7 days.

[00146] In some of any embodiments, the polynucleotide is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10,000 nucleotides in length, or any value between any of the above.

[00147] In some of any modalities, the polynucleotide is between or about 2,500, and of or about 5,000 nucleotides, of or about 3,500 and of or about 4,500 nucleotides, or of or about 3,750 nucleotides and of or about 4,250 nucleotides in length.

[00148] Also supplied are cells generated using any of the methods described in this document. In some respects, also provided are compositions comprising such cells. Also supplied are kits and articles of manufacture for use in carrying out any of the methods provided in this document. Also provided are treatment methods and therapeutic uses, such as therapeutic uses in the treatment of a disease or disorder that involves administration of any of the cells or compositions that contain cells described in this document. Brief Description of the Figures

[00149] FIGS. 1A and 1B shows histograms showing cell surface staining of CD8, Vbeta22 (specific staining

recombinant TCR co; FIG. 1A) and MHC-peptide tetramer complexed with antigen peptide (HPV 16 E7 (11-19); designated "HPV E7 (11)") recognized by the recombinant TCR (FIG. 1B), as assessed by flow cytometry, in T cells subjected to gene knockout encoding endogenous TCR (TRAC and TRBC) and modified to express an exemplary recombinant anti-HPV 16 E7 T cell receptor (TCR) using various constructs for HDR-directed expression and integration at the TRAC locus : total sequences encoding recombinant TCRα and TCRβ polynucleotides linked to the EF1α promoter ("SV40 pA EF1α E7 TCR") or MND ("SV40 pA MND E7 TCR") or encoding a total sequence of the recombinant TCRβ chain and sequence partial recombinant TCRα chain for structural integration with the exogenous TRAC gene exon 1 sequence linked to the MND promoter ("MND E7 TCR 3 'arm") or the endogenous TRAC promoter via a ribosome jump sequence Upstream P2A ("P2A arm E7 TCR 3 '"), integrated in the locus TRAC. T cells submitted to transfection by simulation ("Mock") were evaluated as controls.

[00150] FIG. 2 shows a graph depicting the results of a T cell cytolytic assay expressing an exemplary recombinant TCR co-cultured with target cells in a 5: 1 effector to target ratio (E: T), as assessed by lysis of NucLight Red-labeled target cells over time, in T cells subjected to gene knockout encoding endogenous TCR (TRAC and TRBC) and modified to express an exemplary recombinant anti-HPV 16 E7 T cell receptor (TCR) using various constructs for expression and integration directed by HDR in the TRAC locus: total sequences encoding polynucleotides of recombinant TCRα and TCRβ chains linked to the EF1α ("SV40 pA EF1α E7 TCR") or MND ("SV40 pA MND E7 TCR") or that encodes a total sequence of the recom TCRβ chain -

and partial sequence of the recombinant TCRα chain for integration into structure with an exon sequence of the endogenous TRAC gene, linked to the MND promoter ("MND E7 TCR 3 'arm") or the endogenous TRAC promoter via a P2A sequence of ribosome jump ("arm P2A E7 TCR 3 '"), integrated in the TRAC locus. T cells subjected to transfection by simulation ("Simulation") and cells expressing a TCR reference ("Ref E7 mouse") were used as controls.

[00151] FIGS. 3A-3B depict the results for integration at various time points for the various homology arm lengths tested, as assessed by changes in GFP standards at 24, 48 and 72 hours (FIG. 3A), and at 96 hours or 7 days (FIG. 3B) after transduction with AAV preparations containing the HDR polynucleotides.

[00152] FIGS. 4A-4B depict the change in integration ratio for HDR using the various homology arm lengths in 24, 48, 72 and 96 hours or 7 days for four different donors, Donor 1 and 2 (FIG. 4A) and Donor 3 and 4 (FIG. 4B). Detailed Description

[00153] Provided herein are genetically modified immune cells expressing a recombinant receptor, such as a recombinant T cell receptor (TCR). In some modalities, the genetically modified immune cells provided are T cells that comprise a modified T cell receptor alpha (TRAC) constant locus or a modified T cell receptor (TRBC) beta constant locus that comprises a nucleic acid that encodes a recombinant TCR or part of it. In some embodiments, the nucleic acid sequence is or includes a fusion of a transgene and an open reading frame, or a partial sequence thereof, of an endogenous TRAC locus that encodes a

alpha T cell receptor (TCRα) constant domain. In certain modalities, the transgene encodes a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), where the portion is less than the length TCRα chain total length, for example, a full-length native TCRα chain. In particular embodiments, the nucleic acid sequence is or includes a fusion of a transgene and an open reading frame, or a partial sequence thereof, of an endogenous TRBC locus that encodes a constant T cell beta receptor (TCRβ) domain . In certain embodiments, the transgene encodes a TCRα chain and a part of a TCRβ chain, and a part of a TCRβ chain, where the portion is less than a full-length TCRβ chain, for example, a full-length TCRβ chain . Also provided are related cell compositions, methods for generating or producing the modified cells, nucleic acids and kits for use in generating the modified cells described in this document.

[00154] T cell-based therapies, such as adoptive T cell therapies (including those involving administration of modified cells expressing recombinant TCRs specific to a disease or disorder of interest) may be effective in the treatment cancer and other diseases and disorders. In certain contexts, methods available for generating modified cells for adoptive cell therapy may not always be entirely satisfactory. In some contexts, optimal efficacy may depend on the ability of administered cells to express recombinant TCR, and for uniform, homogeneous and / or consistent expression of receptors between cells, such as a population of immune cells and / or cells in a therapeutic cell composition.

[00155] In certain aspects, the methods currently available, for example, viral transduction, are not entirely satisfactory. For example, in some respects, the efficiency of the expression of the corresponding TCR is limited between certain cells or certain cell populations that are modified using currently available methods. In some cases, recombinant TCR is only expressed in certain cells among a population of cells, and the level of expression of the recombinant TCR can vary widely between cells in the population. In particular aspects, the level of expression of the recombinant TCR can be difficult to predict, control and / or regulate. In some aspects, random integration of a nucleic acid sequence encoding the recombinant TCR into the cell's genome may, in some cases, result in adverse and / or unwanted effects due to the integration of a nucleic acid sequence into a cell. unwanted location in the genome, for example, in an essential gene or a critical gene in regulating cell activity. In some cases, random or semi-random integration of a nucleic acid sequence encoding the receptor can result in antigen binding or subidial, varied, unregulated and / or uncontrolled expression, oncogenic transformation and transcriptional silencing of the nucleic acid sequence, depending on the integration site and / or nucleic acid sequence copy number. In some respects, insertion of the recombinant TCR into the T cell genome may result in incorrect pairing between the recombinant TCR chains and the native TCR chains, thus reducing the amount of recombinant function TCRs expressed by cells.

[00156] In some aspects, variable integration of the sequences encoding the recombinant receptor can result in inconsistent expression, number of variable copies of nucleic acids, possible insertional mutagenesis and / or variability in receptor expression and / or genetic disruption within the cell composition, such as a therapeutic cell composition. In some respects, the use of certain

mined random integration vectors, such as certain lenticular vectors, requires the performance of a competent replication lentivirus assay (RCL).

[00157] In some embodiments, targeted genetic disruption of one or more of the endogenous TCR gene locus may lead to a reduction in risk or change in incorrect pairing between the modified or recombinant TCR and endogenous TCR chains. The erroneously paired TCR can, in some respects, create a new TCR that can potentially result in an increased risk of recognition of unwanted or unwanted antigen and / or side effects, and / or could reduce the expression levels of the Desired modified or recombinant TCR. In some respects, reducing or preventing the expression of endogenous TCR can increase the expression of modified or recombinant TCR in a T cell or T cell compositions compared to cells in which TCR expression is not reduced or prevented. In some modalities, the expression of recombinant TCR can be increased by 1.5 times, 2 times, 3 times, 4 times, 5 times or more. For example, in some cases, the subideal expression of a modified or recombinant TCR may occur due to competition with an endogenous TCR and / or with TCRs having improperly matched chains, for signaling domains such as, signaling molecules from Invariant CD3 that are involved in allowing the expression of the complex on the cell surface. In some respects, methods currently available for the release of transgenes, for example, which encode recombinant receptors, such as recombinant TCRs, may show inefficient integration and / or reduced expression of recombinant receptors. In some aspects, the efficiency of integration and / or expression of the matching receptor within a population may be low and / or varied.

[00158] In some aspects, the development of a human TCR

and / or fully human recombinant presents technical challenges. For example, in some respects, a recombinant humanized and / or fully human TCR competes with endogenous TCR complexes and may form mismatches with endogenous TCRα or TCRβ chains, which may, in certain respects, reduce recombinant TCR signaling, activity, and / or expression. One method to address these challenges has been to design recombinant TCRs with constant mouse domains to avoid incorrect pairs with endogenous human TCRα or TCRβ chains. However, the use of recombinant TCRs with mouse sequences can, in some aspects, present a risk for an immune response. The supplied polynucleotides, reagents, manufacturing articles, kits and methods address these challenges by inserting a portion of a recombinant TCR in structure into an endogenous TCR gene, resulting in a modified locus that encodes a complete recombinant TCR. In particular aspects, this insertion serves to interrupt the expression of the endogenous TCR gene while allowing the expression of a recombinant and / or fully humanized human TCR, reducing the likelihood of competition from or wrong pairs with endogenous TCR chains .

[00159] In some respects, the modalities provided also allow the use of smaller nucleic acid sequence fragments for modification compared to existing methods, using a portion or all open reading structure sequences of the endogenous gene that encodes a constant domain of TCRα or TCRβ, to encode the TCRα or TCRβ chain, or portion thereof, of the recombinant TCR. In some respects, the modalities provided may allow accommodation of larger homology arms compared to conventional modalities that require the entire length of the recombinant TCR in the introduced polynucleotide,

and / or allow for the accommodation of nucleic acid sequences encoding additional molecules, such as the length requirement for nucleic acid sequences encoding the recombinant TCR or a portion thereof is reduced. In some respects, generation, release of nucleic acid sequences, for example, transgenic sequence, and / or homology-directed repair (HDR) targeting efficiency, can be facilitated or improved using the modalities provided.

[00160] In some forms, provided herein are methods of generating or producing genetically modified cells comprising a modified TRAC or TRBC locus wherein the modified TRAC or TRBC locus includes nucleic acid sequences encoding a recombinant TCR. In some respects, the modified TRAC or TRBC locus in the genetically modified cell comprises a transgene sequence (also referred to herein as exogenous or heterologous nucleic acid sequences) that encodes a portion of a recombinant TCR, integrated into a TRAC locus or endogenous TRBC, which normally encodes a constant domain of TCRα or TCRβ. In some modalities, the methods involve the induction of a target genetic disruption and homology-dependent repair (HDR), using standard polynucleotides that contain the transgene that encodes a portion of the recombinant TCR, thus aiming at the integration of the transgene into the locus TRAC or TRBC. Also provided are cells and cell compositions generated by methods.

[00161] In some embodiments, the supplied polynucleotides, transgenes, and / or vectors, when released in immune cells, result in the expression of recombinant TCRs that can modulate T cell activity and, in some cases, can modulate differentiation of T cells or homeostasis. The resulting genetically modified cells or cellular compositions can be used in methods of adoptive cell therapy.

[00162] Thus, the modalities provided can facilitate the production of modified cells that exhibit improved expression, expression function and uniformity and / or other desired characteristics or properties and, ultimately, greater effectiveness. The patterns provided can also reduce the length of polynucleotides, transgenes, and / or vectors needed to release recombinant TCR to cells, for example, to allow sufficient space to package additional elements and / or transgenes within the same vector, for example, viral vector.

[00163] Also provided are methods of modifying, preparing and producing modified cells, kits and devices for generating or producing modified cells. Polynucleotides are provided, for example, viral vectors that contain a nucleic acid sequence that encodes a portion of the chimeric receptor, and methods for introducing such polynucleotides into cells, such as by transduction or by physical release, such as, electroporation. Also provided are compositions containing the modified cells, methods, kits and devices for administering the cells and compositions to individuals, such as for adoptive cell therapy. In some respects, cells are isolated from an individual, modified, and administered to the same individual. In other respects, they are isolated from an individual, modified, and administered to another individual.

[00164] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication were individually incorporated by reference. If a definition presented in this document is contrary to or otherwise inconsistent with a definition presented in patents, applications, published applications

and other publications that are incorporated into this document by reference, the definition in this document presented prevails over the definition that is incorporated in this document by reference.

[00165] The section titles in this document used are for organizational purposes only and should not be interpreted as limiting the subject. I. METHODS FOR PRODUCING EXPRESSING CELLS

A RECOMBINANT RECEIVER FOR HOMOLOGY REPAIR (HDR)

[00166] Provided in this document are methods of producing a genetically modified immune cell, for example, a genetically modified T cell for adoptive cell therapy, related compositions, methods, uses and kits and used manufacturing articles to carry out the methods. Also provided are genetically modified immune cells expressing a recombinant receptor, such as a recombinant T cell receptor (TCR) and compositions containing such cells, including genetically modified immune cells produced by any of the methods provided. Immune cells are generally modified to express a recombinant molecule, such as a recombinant receptor, for example, a recombinant T cell receptor (TCR). In some aspects, the modalities provided specifically involve targeting nucleic acid sequences that encode a part of the matching receptor, for example, a TCR, to a particular locus, for example, at one or more target sites within the locus of endogenous TCR gene. In some embodiments, the nucleic acid sequences are integrated in structure within the TCR gene locus to produce a modified gene locus that encodes the total recombinant TCR.

[00167] In some embodiments, methods are provided to produce a genetically modified immune cell, for example, a genetically modified T cell for adoptive cell therapy. In some embodiments, the methods provided involve the introduction into an immune cell of one or more agents capable of inducing a genetic disruption of one or more target sites (also known as "target position," "target DNA sequence" or "target location ") within a gene encoding a domain or region of a T cell alpha receptor (TCRα) chain and / or one or more gene (s) encoding a domain or region of a T cell beta receptor chain (( TCRβ) (also referred to as "one or more agents" or "agent (s) with reference to aspects of the methods provided); and introducing into the immune cell a polynucleotide, for example, a standard polynucleotide, which comprises a transgene that encodes a recombinant receptor or strand thereof, wherein the transgene encoding a recombinant receptor or strand thereof is targeted at or near one of at least one target site by means of homology-directed repair (HDR).

[00168] In some embodiments, provided in this document are methods of generating or producing genetically modified cells comprising a modified TRAC or TRBC locus in which the modified TRAC or TRBC locus includes nucleic acid sequences encoding a recombinant TCR . In some respects, the modified TRAC or TRBC locus in the genetically modified cell comprises a transgene sequence (also referred to herein as exogenous or heterologous nucleic acid sequences) that encodes a part of a recombinant TCR, integrated into an endogenous TRAC or TRBC locus, which normally encodes a constant domain of TCRα or TCRβ. In some modalities, the methods involve inducing a target genetic disruption and repair

pending homology (HDR), using standard polynucleotides that contain the transgene that encodes a portion of the recombinant TCR, thus targeting integration of the transgene into the TRAC or TRBC locus. Also provided are cells and cell compositions generated by the methods.

[00169] In some embodiments, the transgene sequence that encodes a portion of the recombinant TCR contains a nucleotide sequence that encodes a TCRβ chain and a portion of a TCRα chain. In some embodiments, the portion of the TCRα chain encoded by the transgenic sequence comprises less than a total length of the TCRα chain. In particular embodiments, the TCRα chain portion contains a TCRα variable domain and a portion of a TCRα constant domain that is less than a full-length TCR constant domain, for example, a full-length native TCRα constant domain , or does not contain a sequence that encodes a constant domain TCRα. In some respects, after integration of the transgene sequence into the endogenous TRAC locus, the resulting modified TRAC locus encodes a matching TCR receptor, encoded by a transgene fusion, targeted by HDR, and an open reading frame or a partial sequence of that of an endogenous TRAC locus. In some embodiments, the encoded recombinant TCR contains a TCRα chain, for example, a functional TCRα chain that is capable of binding to a TCRβ chain.

[00170] In particular embodiments, the transgene sequence that encodes a portion of the recombinant TCR contains a nucleotide sequence that encodes a TCRα chain and / or a portion of a TCRβ chain. In some embodiments, the portion of the TCRβ chain encoded by the transgene sequences is or includes less than a total length of the TCRβ chain. In particular modalities

res, the portion of the TCRβ chain contains a variable domain of TCRβ and a portion of a constant domain of TCRβ that is less than the full-length TCR constant domain, for example, a full-length native TCRα constant domain, or it does not contain a sequence that encodes the constant domain of TCRβ. In some respects, after integrating the transgene sequence into the endogenous TRBC locus, for example, a TRBC1 and / or TRBC2 locus, the resulting modified TRBC locus encodes a matching TCR receptor, encoded by a fusion of the transgene, targeted by HDR, and an open reading frame or partial sequence thereof from an endogenous TRBC locus. In some embodiments, the encoded recombinant TCR contains a TCRβ chain, for example, a TCRβ functional chain that is capable of binding to a TCRα chain.

[00171] In some embodiments, the polynucleotide, for example, the standard polynucleotide, comprises a nucleic acid sequence that encodes a fraction and / or a portion of a recombinant receptor or chain thereof, for example, a TCR recombinant or a chain thereof. In certain embodiments, the nucleic acid sequence is targeted at a target site (s) that is within a gene locus that encodes an endogenous receptor, for example, an endogenous TCR gene. In certain embodiments, the nucleic acid sequence is targeted for integration into structure within the endogenous gene locus. In particular embodiments, integration in structure results in a coding sequence for the recombinant receptor that contains the nucleic acid sequence that encodes the portion and / or fragment of the recombinant receptor in structure with the portion and / or fragment of the locus of the gene encoding the remaining portion and / or fragment of the receptor, such as to integrate exogenous and endogenous nucleic acid sequences to arrive at a coding sequence that encodes a complete, whole and / or recombinant receptor - total length. In certain embodiments, integration genetically disrupts the expression of the endogenous receptor encoded by the gene at the target site. In particular embodiments, the transgene that encodes the recombinant receptor portion is targeted within the gene locus by means of HDR.

[00172] In particular embodiments, the recombinant receptor is a recombinant TCR or strand thereof that contains one or more variable domains and one or more constant domains. In particular modalities, the transgene encodes the portion and / or fraction of the recombinant TCR that does not include a constant TCR domain, and the transgene is integrated in structure with the sequence, for example, genomic DNA sequence, that encodes an endogenous TCR constant domain. In certain embodiments, the integration results in a coding sequence that encodes the complete, whole, and / or full-length recombinant TCR or its strand. In some embodiments, the coding sequence contains the transgene sequence that encodes the portion or fragment of the TCR or strand thereof and an endogenous sequence that encodes the constant domain of the endogenous TCR.

[00173] In some embodiments, the transgene encodes a portion and / or a fragment of the recombinant receptor that includes a portion and / or a fraction of a constant domain, for example, a portion or fragment of the constant domain that is completely or partially identical to an endogenous TCR constant domain. In some embodiments, the encoded transgene is integrated in structure with the sequence, for example, genomic DNA sequence, which co-complicates a portion and / or fragment of the endogenous TCR constant domain that is not encoded by the transgene. In particular modalities, the integration results in a coding sequence that encodes

it contains the complete, whole and / or full length recombinant TCR or strand thereof and contains the transgene sequence and the endogenous sequence encoding an endogenous portion or fragment of the constant domain of the TCR.

[00174] In some embodiments, the methods provided involve an introduction into an immune cell of one or more agents, in which each or more agent is independent capable of inducing a genetic disruption of a target site within a constant alpha receptor gene T cell (TRAC) and / or a T cell beta receptor constant gene (TRBC), thereby inducing a genetic disruption of at least one target site; and introducing into the immune cell a standard polynucleotide that comprises a transgene encoding a recombinant T cell receptor (TCR) or an antigen-binding fragment thereof or a chain thereof, wherein the transgene encoding the recombinant TCR or fragment of binding to the antigen or its chain is targeted for integration into or near at least one target site by means of repair directed to homology (HDR). In particular embodiments, integration at or near the target site is structured with a portion of the coding sequence of the TRAC or TRBC gene, such as, for example, a portion of the coding sequence downstream, for example, immediately downstream and / or 3 'adjacent to the target site.

[00175] In some embodiments, one of at least one target site is in the alpha T cell receptor constant (TRAC) gene. In some embodiments, one of at least one of the target sites is in a T cell beta receptor constant gene (TRBC1) or T cell beta receptor constant gene (TRBC2). In some modalities, one or more target sites are in a TRAC gene and one or both of a TRBC1 and TRBC2 gene.

[00176] In some embodiments, the methods provided involve an introduction into an immune cell having a genetic disruption of one or more target sites within a gene encoding a domain or region of an alpha T cell receptor (TCRα) chain and / or one or more genes encoding a domain or region of a T cell beta receptor (TCRβ) chain, a standard polynucleotide that comprises a transgene encoding a recombinant receptor, where the transgene encoding a receptor recombinant or a strand thereof is targeted at or near one of at least one target site by means of HDR.

[00177] In embodiments, the term "introduction" encompasses a variety of methods of introducing DNA into a cell, whether in vitro or in vivo, such methods including transformation, transduction, transfection (e.g., electroporation), and infection. Vectors are useful for introducing molecules that encode DNA into cells. Possible vectors include plasmid vectors and viral vectors. Viral vectors include retroviral vectors, lentiviral vectors or other vectors, such as adenoviral vectors or adeno-associated vectors. Methods, such as electroporation, can also be used to introduce or release protein or ribonucleoprotein (RNP), for example, which contains the Cas9 protein in the complex with a target gRNA, for cells of interest.

[00178] In some cases, the modalities provided in this document involve targeted genetic disruption, for example, DNA breakage, in one or more of the endogenous TCR gene locus (such as, the endogenous genes that encode the TCRα and / or chains TCRβ) by gene editing techniques, combined with targeted nucleic acid knockout encoding the recombinant receptor (such as, a TCR or a recombinant CAR) by homology-directed repair (HDR). In some modalities, the HDR step requires a break, for example, a double strand break, in the DNA at the target genomic location. In some modalities, DNA breakdown occurs as a result of a gene editing step, for example, DNA breaks generated by targeted nucleases used in gene editing.

[00179] In some modalities, the modalities involve generating a targeted DNA break using methods of editing genes and / or targeted nucleases, followed by HDR based on one or more standard polynucleotides, for example, standard polynucleotides that contain sequences of homology and one or more transgenes, for example, nucleic acids encoding a recombinant receptor or a strand thereof and / or other exogenous or recombinant nucleic acids, specifically to target and integrate the nucleic acid sequences encoding the recombinant receptor or a strand of the same and / or other exogenous or recombinant nucleic acids at or near DNA break.

[00180] In some embodiments, the targeted genetic disruption and targeted integration of nucleic acids encoding the corresponding HDR receptor occurs at one or more target sites (also known as "target position", "target DNA sequence" or "target location") of endogenous genes encoding one or more domains, regions and / or chains of endogenous T cell receptor (TCR). In some embodiments, the targeted genetic disruption is induced in the TCRα gene. In some modalities, the targeted genetic disruption is induced in the TCRβ gene. In some modalities, the targeted genetic disruption is induced in the endogenous TCRα gene and the endogenous TCRβ gene. Endogenous TCR genes can include one or more of the gene that co-replicates TCRα constant domain (encoded by TRAC in humans) and / or TCRβ constant domain (encoded by TRBC1 or TRBC2 in humans). In particular embodiments, the polynucleotide, for example, standard polynucleotide, contains a sequence of nucleic acid

cell encoding a portion and / or fragment of a recombinant TCR that contains a portion and / or a fragment of a TCRα chain. In some embodiments, the TCRα chain portion and / or fragment contains a full, full and / or full-length TCRα variable domain and a portion and / or a fraction of the constant TCRα domain. In certain embodiments, the nucleic acid sequence is integrated in structure at a target site within a TRAC gene. In some embodiments, integration in structure results in a coding sequence that encodes a complete, whole, and / or full-length recombinant TCR or chain. In particular modalities, the complete, whole and / or full length recombinant receptor contains an entire, complete and / or full length TCRα chain. In some embodiments, the full, whole, and / or full-length recombinant receptor contains a full, full, and / or full-length TCRα constant domain.

[00181] In certain embodiments, the polynucleotide, for example, standard polynucleotide, encodes a portion and / or fragment of a recombinant TCR or strand thereof that contains a portion or fragment of a TCRβ chain. In some embodiments, the portion and / or fragment of the TCRβ chain contains a portion and / or a fragment of a constant domain of TCRβ. In certain embodiments, the transgene is integrated in structure at a target site within a TRBC gene, for example, TRBC1 and / or TRBC2. In some modalities, integration in structure results in a coding sequence that encodes a complete, whole and / or full length recombinant TCR or chain. In particular embodiments, the whole, complete and / or full-length recombinant receptor contains a complete, whole, and / or full-length TCRβ chain. In some embodiments, the whole, full, and / or full-length recombinant receptor contains a full, full, and / or full-length TCRβ constant domain.

[00182] In some embodiments, a standard polynucleotide is introduced into the modified cell, prior to, simultaneously with, or subsequent to the introduction of an agent (s) capable of inducing a target genetic disruption. In the presence of a target genetic disruption, for example, DNA breakdown, the standard polynucleotide can be used as a DNA repair pattern, to effectively copy and integrate the transgene, for example, nucleic acid sequences encoding a recombinant receptor, at or near the site of an HDR-targeted genetic disruption, based on the homology between the endogenous gene sequence involving the target site and the 5 'and / or 3' homology arms included in the standard polynucleotide.

[00183] In some modalities, the steps of editing genes and HDR are performed simultaneously and / or in an experimental reaction. In some modalities, the steps of editing genes and HDR are carried out consecutively or sequentially, in one or several consecutive experimental reactions. In some modalities, the steps of editing genes and HDR are carried out in separate experimental reactions, simultaneously or at different times.

[00184] Immune cells may include a population of cells that contain T cells. Such cells may be cells that were obtained from an individual, such as, obtained from a sample of peripheral blood mononuclear cells (PBMC), a sample of unfractionated T cells, a sample of lymphocytes, a sample of white blood cells, an apheresis product, or leukapheresis product. In some modalities, T cells can be separated or selected to enrich T cells in the population using positive or negative selection and enrichment methods. In some modalities, the population contains CD4 +, CD8 + or CD4 + and CD8 + T cells. In some embodiments, the step of introducing the standard polynucleotide and the step of introducing the agent (eg Cas9 / gRNA RNP) can occur simultaneously or sequentially in any order. In particular modalities, the standard polynucleotide is introduced into the immune cells after the induction of the genetic disruption by the agent introduction step (for example, Cas9 / gRNA RNP). In some embodiments, before, during and / or subsequent to the introduction of the standard polynucleotide and one or more agents (for example, Cas9 / RNP gRNA), cells are cultured or incubated under conditions to stimulate expansion and / or cell proliferation.

[00185] In particular modalities of the methods provided, the introduction of the standard polynucleotide is carried out after the introduction of one or more agents capable of inducing a genetic disruption. Any method for introducing said one or more agents can be used as described, depending on the agent (s) used to induce genetic disruption. In some respects, disruption is accomplished by editing the gene, such as, using an RNA-guided nuclease, such as a regularly interleaved short palindromic nucleic acid (CRISPR) -Cas system, such as the CRISPR-Cas9 system, specific to the TRAC or TRBC locus being interrupted. In some embodiments, an agent that contains a Cas9 and an RNA guide (gRNA) that contains a targeting domain, which targets a region of the TRAC or TRBC locus, is introduced into the cell. In some embodiments, the agent is or comprises a Cas9 and gRNA ribonucleoprotein complex (RNP) that contains the targeting domain targeted by TRAC / TRBC (Cas9 / RNP gRNA). In some embodiments, the introduction includes contact of the agent or portion of it with the cells, in vitro, which may include culturing or incubating the cell and the agent for up to 24, 36 or 48 hours or 3, 4, 5, 6, 7, or 8 days. In some embodiments, the introduction may also include releasing the agent into the cells. In several modalities

However, the methods, compositions and cells according to the present invention use the direct release of Cas9 ribonucleoprotein (RNP) and gRNA complexes to cells, for example, by electroporation. In some embodiments, RNP complexes include a gRNA that has been modified to include a 3 'poly-A tail and a 5' anti-reverse Cap analog buffer (ARCA). In some cases, the electroporation of cells to be modified includes cold shock to the cells, for example, at 32 ° C after electroporation of the cells and before plating.

[00186] In these aspects of the methods provided, a standard polynucleotide is introduced into the cells after introduction with said one or more agents, such as Cas9 / gRNA RNP, for example, which was introduced by means of electroporation. In some modalities, the standard polynucleotide is introduced immediately after the introduction of one or more agents capable of inducing a genetic disruption. In some embodiments, the standard polynucleotide is introduced into cells within or about 30 seconds, within or about 1 minute, within or about 2 minutes, within or about 3 minutes, within or about 4 minutes, within or about 5 minutes, within or about 6 minutes, within or about 8 minutes, within or about 9 minutes, within or about 10 minutes, within or about 15 minutes, in or about 20 minutes, in or about 30 minutes, in or about 40 minutes, in or about 50 minutes, in or about 60 minutes, in or about 90 minutes , within or about 2 hours, within or about 3 hours or within or about 4 hours after the introduction of one or more agents capable of inducing a genetic disruption. In some embodiments, the standard polynucleotide is introduced into the cells between or about 15 minutes and or about 4 hours after the introduction of said one or more agents,

such as between or about 15 minutes and or about 3 hours, between and about 15 minutes and about or about 2 hours, between or about 15 minutes and about or about 1 hour, between or about 15 minutes and about or about 30 minutes, between about or about 30 minutes and about or about 4 hours, between about or about 30 minutes and about or about 3 hours, between about or about 30 minutes and about or about 2 hours, between or about 30 minutes and about or about 1 hour, between or about 1 hour and about or about 4 hours, between about or about 1 hour and about or about 3 hours, between about or about 1 hour and about or about 2 hours, between about or about 2 hours and about or about 4 hours, between about or about 2 hours and about or about 3 hours or between or about 3 hours and about 4 hours. In some embodiments, the standard polynucleotide is introduced into cells at or about 2 hours after the introduction of one or more agents, such as Cas9 / gRNA RNP, for example, which was introduced by means of electroporation.

[00187] Any method for introducing the standard polynucleotide can be used as described, depending on the methods used to release the standard polynucleotide to cells. Exemplary methods include those for transferring nucleic acids that encode the receptor, including via viral, for example, retroviral or lentiviral, transduction, transposons and electroporation. In particular modalities, viral transduction methods are used. In some modalities, standard polynucleotides can be transferred or introduced into cells by recombinant infectious virus particles, such as vectors derived from simian virus 40 (SV40), adenovirus, adenoassociated viruses (AAV). In some embodiments, recombinant nucleic acids are transferred to T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, for example, Koste et al. (2014) Gene Therapy April 3, 2014 doi: 10.1038 / gt.2014.25; Carlens et al. (2000) Exp

Hematol 28 (10): 1137-46; Alonso-Camino et al. (2013) Mol Ther Nucl Acids 2, e93; Park et al., Trends Biotechnol. November 29, 2011 (11): 550–557. In particular embodiments, the viral vector is an AAV, such as an AAV2 or an AAV6.

[00188] In some embodiments, before, during or subsequent to contact of the agent with the cells and / or before, during or subsequent to effecting the release (for example, electroporation), the methods provided include incubating the cells in the presence of a cytokine, a stimulating agent and / or an agent that is capable of inducing proliferation, stimulation or activation of immune cells (for example, T cells). In some embodiments, at least a portion of the incubation is in the presence of a stimulating agent that is or comprises an antibody specific for CD3, an antibody specific for CD28 and / or a cytokine, such as anti-CD3 / anti-CD28. In some embodiments, at least a portion of the incubation is in the presence of a cytokine, such as one or more of the recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. In some embodiments, the incubation is for up to 8 days before or after the introduction with said one or more agents, such as Cas9 / gRNA RNP, for example, by means of electroporation, and standard polynucleotide, such as, up to 24 hours, 36 hours or 48 hours or 3, 4, 5, 6, 7 or 8 days.

[00189] In some embodiments, the method includes activation or stimulation of cells with a stimulating agent (for example, anti-CD3 / anti-CD28 antibodies) before the introduction of the agent, for example, Cas9 / gRNA RNP, and the standard polynucleotide. In some modalities, incubation in the presence of a stimulating agent (for example, anti-CD3 / anti-CD28) is for 6 hours to 96 hours, such as 24-48 hours or 24-36 hours before introduction with said one or more agents, such as Cas9 / gRNA RNP, for example, by means of electroporation. In some embodiments, incubation with stimulating agents may also include the presence of a cytokine, such as one or more of recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. In some embodiments, incubation is performed in the presence of recombinant cytokines, such as IL-2 (for example, 1 U / ml to 500 U / ml, such as, 10 U / ml to 200 U / ml, for example by less or about 50 U / ml or 100 U / ml), IL-7 (for example, 0.5 ng / ml to 50 ng / ml, such as, 1 ng / ml to 20 ng / ml, for example, at least or about 5 ng / ml or 10 ng / ml) or IL-15 (for example, 0.1 ng / ml to 50 ng / ml, such as, 0.5 ng / ml to 25 ng / ml, for example, at least or about 1 ng / ml or 5 ng / ml). In some embodiments, stimulating agents (for example, anti-CD3 / anti-CD28 antibodies) are washed or removed from cells before introducing or releasing into cells cells capable of introducing a Cas9 / gRNA RNP and / or polynucleotide rupture pattern. In some modalities, before the agents are introduced, the cells are rested, for example, by removing any stimulating or activating agent. In some embodiments, before introducing the agents, the stimulating or activating agent and / or cytokines are not removed.

[00190] In some embodiments, subsequent to the introduction of the agents, for example, Cas9 / gRNA, and / or the standard polynucleotide, cells are incubated, cultured (cultured) or cultured (cultured) in the presence of recombinant cytokines, such as, one or more of recombinant IL-2, recombinant IL-7 and / or recombinant IL-15. In some embodiments, incubation is performed in the presence of recombinant cytokines, such as IL-2 (for example, 1 U / ml to 500 U / ml, such as, 10 U / ml to 200 U / ml, for example example, at least or about 50 U / mL or 100 U / mL), IL-7 (for example, 0.5 ng / mL to 50 ng / mL, such as 1 ng / mL to 20 ng / mL, for example, at least or about 5 ng / mL or 10 ng / mL) or IL-15 (for example, 0.1 ng / mL to 50 ng / mL, such as, 0.5 ng / mL to 25 ng / ml, for example, at least or about 1 ng / ml or 5 ng / ml). The cells can be incubated or cultured under conditions to induce cell proliferation or expansion. In some embodiments, cells can be incubated or cultured until a limited number of cells are activated for collection, for example, a therapeutically effective dose.

[00191] In some modalities, the incubation during any portion of the process or the whole process can be at a temperature of 30º C ± 2º C to 39º C ± 2º C, such as, at least or about at least 30º C ± 2º C, 32º C ± 2º C, 34º C ± 2º C or 37º C ± 2º C. In some modalities, at least a portion of the incubation is at 30º C ± 2º C and at least a portion of the incubation is at 37º C ± 2º Ç.

[00192] In some embodiments, after targeted integration, the nucleic acid sequence present in the modified TRAC or TRBC locus comprises a fusion of an HDR-targeted transgene, and an open reading frame or partial sequence thereof of an endogenous TRAC or TRBC locus. In some respects, the nucleic acid sequence present in the modified TRAC or TRBC locus comprises a transgene that is integrated with an endogenous TRAC or TRBC locus that contains an open reading frame encoding a TCRα or TCRβ constant domain . In some respects, and after targeted integration or fusion, for example, fusion into structure, a portion of the exogenous sequence and a portion of the open reading frame at the endogenous TRAC or TRBC locus together encode a recombinant TCRα or TCRβ chain . Thus, the provided modalities use a portion or all sequences of open reading structure of endogenous TRAC or TRBC locus to encode the entire TCRα or TCRβ chain of the recombinant TCR.

[00193] In particular embodiments, the nucleic acid sequence present in the modified TRAC locus is or includes a fusion of an HDR-targeted transgene, and an open reading frame or a partial sequence thereof from an endogenous TRAC locus. In certain embodiments, the nucleic acid sequence present in the modified TRAC locus comprises a transgene that is integrated into an edogenous TRAC locus that contains an open reading frame encoding a constant domain of TCRα. In particular embodiments, after targeted integration or fusion, for example, structure fusion, a portion of the exogenous sequence and a portion of the open reading frame at the endogenous TRAC locus together encode a recombinant TCRα chain and / or a protein that contains a constant domain TCRα. In particular embodiments, a TCRα chain and / or a protein that contains a TCRα constant domain contains a functional constant domain TCRα, for example, a constant domain TCRα that is capable of binding to a TCRβ chain.

[00194] In some embodiments, the nucleic acid sequence present in the modified TRBC locus, for example, a modified TRBC1 and / or TRBC2 locus, is or includes a fusion of an HDR-targeted transgene, and a reading frame open or a partial sequence thereof of an endogenous TRBC locus. In certain embodiments, the nucleic acid sequence present in the modified TRBC locus comprises a transgene that is integrated into an edogenous TRAC locus that contains an open reading frame encoding a constant domain of TCRβ. In particular embodiments, after targeted integration or fusion, for example, structure fusion, a portion of the exogenous sequence and a portion of the open reading frame at the endogenous TRBC locus together encode a recombinant TCRβ chain and / or a protein that contains a constant domain of TCRβ. In particular embodiments, a TCRβ chain and / or a protein that contains a TCRβ constant domain contains a functional TCRβ constant domain, for example, a TCRβ constant domain that is capable of binding to a TCRβ chain.

A. Genetic Disruption

[00195] In some embodiments, one or more targeted genetic disruptions are induced in the endogenous TCRα gene and / or the endogenous TCRβ gene. In some embodiments, targeted genetic disruption is induced in one or more of the gene encoding the TCRα constant domain (also known as the TCRα constant region; encoded by TRAC in humans) and / or the TCRβ constant domain (also known as the constant region of TCRβ; encoded by TRBC1 or TRBC2 in humans). In some modalities, targeted genetic disruption is induced at the locus TRAC, TRBC1 and TRBC2. In some modes, the targeted genetic disruption is induced in an intron, for example, a TRAC, TRBC1 or TRBC2 intron. In some modalities, the targeted genetic disruption is induced in an exon, for example, a TRAC, TRBC1 or TRBC2 exon.

[00196] In some modalities, the targeted genetic disruption results in a DNA break or an incision. In some modalities, at the DNA break site, the action of cellular DNA repair mechanisms can result in knockout, insertion, wrong sense or structure mutation, such as a biallelic structure mutation, deletion of all or part of the gene. In some embodiments, the genetic disruption can be targeted to one or more exons of a gene or portion thereof, such as in the first or second exon. In some embodiments, a DNA-binding protein or DNA-binding nucleic acid, which specifically binds to or hybridizes to sequences in a region close to one of at least one target site, is used for targeted disruption. In some respects, in the absence of the standard exogenous polynucleotides for HDR disruption, the targeted genetic disruption results in a deletion, mutation and or insertion into an exon of the gene. In some embodiments, standard polynucleotides, for example, standard polynucleotides that include nucleic acid sequences encoding a recombinant receptor and homology sequences, can be introduced for targeted integration of the recombinant receptor coding sequences at or near the site of the genetic disruption by HDR, such as any integration described in this document, for example, in Section IB

[00197] In some modalities, genetic disruption is carried out by the introduction of one or more agents capable of inducing a genetic disruption. In some embodiments, these agents comprise a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the gene. In some embodiments, the agent comprises several components, such as a fusion protein that comprises a DNA targeting protein and an RNA-guided nuclease or nuclease. In some modalities, agents may target one or more target locations, for example, in a TRAC gene and one or both of a TRBC1 and TRBC2 gene.

[00198] In some modalities, genetic disruption occurs at a target site (also referred to and / or known as "target position", "target DNA sequence" or "target location"). In some modalities, the target site is or includes a site in a target DNA (for example, genomic DNA) that is modified by one or more agents capable of inducing a genetic disruption, for example, a complexed Cas9 molecule with a gRNA that specifies the target site. For example, in some embodiments, the target site may include locations in DNA, for example, in an endogenous TRAC or TRBC locus, where DNA cleavage or breakage occurs. In some respects, integration of nucleic acid sequences by HDR can occur at or near the target site or target sequence. In some embodiments, a target site can be a site between two nucleotides, for example, adjacent nucleotides, in the DNA to which one or more nucleotides are added.

The target site can comprise one or more nucleotides that are altered by a standard polynucleotide. In some embodiments, the target site is within the target sequence (for example, a sequence to which the gRNA binds). In some embodiments, a target site is upstream or downstream of a target sequence.

1. Target sites in Genes that encode Endogenous T Cell Receptor (TCR)

[00199] In some embodiments, there is a targeted genetic disruption in endogenous genes that encode one or more domains, regions and / or chains of the endogenous T cell receptor (TCR). In some embodiments, the genetic disruption is targeted at the locus of endogenous genes that encode TCRα and / or TCRβ. In some embodiments, the genetic disruption is targeted at the gene encoding the TCRα constant domain (TRAC in humans) and / or the TCRβ constant domain (TRBC1 or TRBC2 in humans).

[00200] In some embodiments, a "T cell receptor" or "TCR", including endogenous TCRs, is a molecule that contains a variable α and β chain (also known as TCRα and TCRβ, respectively) or a γ and variable δ (also known as TCR γ and TCR δ, respectively), or antigen-binding portions thereof, which are able to specifically bind to a peptide bound to an MHC molecule. In some modalities, the TCR is in αβ form. Typically, TCRs that exist in αβ and γδ forms are generally structurally similar, but the T cells that express them may have different anatomical locations or functions. Typically, a T cell expresses a type of TCR. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for recognizing antigens linked to molecules of the complex of greater histocompatibility

(MHC).

[00201] In some embodiments, a TCR may contain a variable domain and a constant domain (also known as a constant region), a transmembrane domain and / or a short cytoplasmic tail (see, for example, Janeway et al ., Immunobiology: The Immune System in Health and Disease, 3rd Ed., Current Biology Publications, p. 4:33, 1997). In some embodiments, a TCR chain contains one or more constant domains. For example, the extracellular portion of a given TCR chain (for example, TCRα chain or TCRβ chain) can contain two immunoglobulin-like domains, such as a variable domain (for example, Vα or Vβ; typically amino acids 1 to 116 based on Kabat numbering, Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5th ed.) and a constant domain (for example, α chain or TCR Cα domain, typically positions 117 to 259 of the chain based on Kabat numbering or chain constant domain β or TCR Cβ, typically positions 117 to 295 of the Kabat-based chain) adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two constant domains proximal to the membrane and two variable domains distal to the membrane.

[00202] In some embodiments, the endogenous TCR Cα is encoded by the TRAC gene (IMGT nomenclature). An exemplary nucleotide sequence of the human T cell alpha receptor constant chain (TRAC) gene locus is set out in SEQ ID NO: 1 (NCBI reference sequence: NG_001332.3, TRAC). In some embodiments, the encoded endogenous Cα comprises the amino acid sequence set out in SEQ ID NO: 19 or 24 (UniProTKB adhesion No. P01848 or Genbank adhesion No. CAA26636.1).

[00203] In humans, an exemplary TRAC genomic locus comprises an open reading structure that contains 4 exons and 3 introns. An example of TRAC mRNA transcription can cover the sequence corresponding to the coordinates of chromosome 14:

22.547.506-22.552.154, on the front ribbon, with reference to the version of the human genome GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 1 shows the coordinates of exons and introns of open reading structures and the untranslated regions of the transcription of an exemplary human TRAC locus. Table 1. Coordinates of exons and introns of exemplary human TRAC locus (GRCh38, Chromosome 14, frontal strip). Start (GrCh38) Final (GrCh38) Length 5 'RTU and exon 1 22.547.506 22.547.778 273 Intron 1-2 22.547.779 22.549.637 1.859 Exon 2 22.549.638 22.549.682 45 Intron 2-3 22.549.683 22.550 .556 874 Exon 3 22,550,557 22,550,664 108 Intron 3-4 22,550,665 22,551,604 940 Exon 4 and 3 'RTU 22,551,605 22,552,154 550

[00204] In certain modalities, the genetic disruption is targeted at, near or within a TRAC locus. In particular modalities, the genetic disruption is targeted at, near or within an open reading frame of the TRAC locus. In certain modalities, the genetic disruption is targeted at, near or within an open reading structure that encodes a constant domain TCRα. In some modalities, the genetic disruption is targeted at, near or within a locus having a nucleic acid sequence established in SEQ ID NO: 1, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% of identity string for all or a portion, for example, at or at least 500,

1,000, 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the nucleic acid sequence set out in SEQ ID NO: 1.

[00205] In humans, an exemplary genomic locus of TRBC1 comprises an open reading frame containing 4 exons and 3 introns. An exemplary TRBC1 mRNA transcript can span the sequence corresponding to the coordinates of Chromosome 7: 142,791,694-142,793,368, on the front strip, with reference to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 2 establishes the coordinates of the exons and introns of the open reading structures and the untranslated regions of the transcript of an exemplary human TRBC1 locus. Table 2. Coordinates of exons and introns of the exemplary human TRBC1 locus (GRCh38, Chromosome 7, frontal strip). start (GrCh38) end (GrCh38) length 5 'RTU and exon 1 142,791,694 142,792,080 387 Intron 1-2 142,792,081 142,792,521 441 Exon 2 142,792,522 142,792,539 18 Intron 2-3 142,792,540 142,792 .691 152 Exon 3 142,792,692 142,792,798 107 Intron 3-4 142,792,799 142,793,120 322 Exon 4 and 3 'RTU 142,793,121 142,793,368 248

[00206] In humans, an exemplary genomic locus of TRBC2 comprises an open reading frame containing 4 exons and 3 introns. An exemplary TRBC2 mRNA transcript can span the sequence corresponding to the coordinates of Chromosome 7: 142,801,041-142,802,748, on the front strip, with reference to the human genome version GRCh38 (UCSC Genome Browser on Human Dec. 2013 (GRCh38 / hg38) Assembly). Table 3 establishes the coordinates of the exons and introns of the open reading structures and the untranslated regions of the transcript of an exemplary human TRBC2 locus.

Table 3. Coordinates of exons and introns of exemplary human TRBC2 locus (GRCh38, Chromosome 7, frontal strip). start (GrCh38) end (GrCh38) length 5 'RTU and exon 1 142,801,041 142,801,427 387 Intron 1-2 142,801,428 142,801,943 516 Exon 2 142,801,944 142,801,961 18 Intron 2-3 142,801,962 142,802 .104 143 Exon 3 142,802,105 142,802,211 107 Intron 3-4 142,802,212 142,802,502 291 Exon 4 and 3 'RTU 142,802,503 142,802,748 246

[00207] In some respects, the transgene (for example, exogenous nucleic acid sequences) within the standard polynucleotide can be used to guide the location of target sites and / or homologous arms. In some respects, the site of genetic disruption can be used as a guide to designate standard polynucleotides and / or homology arms used for HDR. In some embodiments, the genetic disruption can be targeted close to a desired site of targeted integration of transgene sequences (for example, which encodes a recombinant TCR or a portion thereof). In some aspects, the target site is within an exon of the open reading structure of the TRAC, TRBC1 and / or TRBC2 locus. In some respects, the target site is within an intron of the open reading structure of the TRAC, TRBC1 and / or TRBC2 locus.

[00208] In some embodiments, the endogenous TCR C is encoded by TRBC1 or TRBC2 genes (IMGT nomenclature). An exemplary nucleotide sequence of the human T cell beta receptor constant chain 1 (TRBC1) locus is set out in SEQ ID NO: 2 (NCBI reference sequence: NG_001333.2, TRBC1); and an exemplary nucleotide sequence of the human T cell beta receptor 2 constant chain locus (TRBC2) is set out in SEQ ID NO: 3 (Reference sequence NCBI: NG_001333.2,

TRBC2). In some embodiments, the encoded C has or comprises an amino acid sequence established in SEQ ID NO: 20, 21 or 25 (Uniprot Adhesion No. P01850, A0A5B9 or A0A0G2JNG9). In some embodiments, the genetic disruption is targeted at, near or within the TRBC1 gene locus. In particular modalities, the genetic disruption is targeted at, near or within an open reading structure of the TRBC1 locus. In certain modalities, genetic disruption is targeted at, near or within an open reading structure that encodes a constant domain of TCRβ. In some modes, the genetic disruption is targeted at, near or within a locus having a nucleic acid sequence established in SEQ ID NO: 2, or a sequence having or at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% of identity sequence for all or a portion, for example, at or at least 500, 1,000, 1,500 , 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the nucleic acid sequence set out in SEQ ID NO:

two.

[00209] In particular modalities, the genetic disruption is targeted at, near or within the TRBC2 locus. In particular modalities, the genetic disruption is targeted at, near or within an open reading structure of the TRBC2 locus. In certain embodiments, the genetic disruption is targeted at, near or within an open reading frame that encodes a constant domain of TCRβ. In some embodiments, the genetic disruption is targeted at, near or within a locus having a nucleic acid sequence established in SEQ ID NO: 3, or a sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% identity string for all or a portion, for example, at or at least 500, 1,000 , 1,500, 2,000, 2,500, 3,000, 3,500, or 4,000 contiguous nucleotides, of the nucleic acid sequence set out in SEQ

ID NO: 3.

[00210] In some embodiments, genetic disruption, for example, DNA breakdown, is targeted at or in close proximity to the beginning of the coding region (for example, the early coding region, for example, within 500bp of the start codon or the remaining coding sequence, for example, downstream of the first 500bp of the start codon). In some embodiments, the genetic disruption, for example, DNA breakdown, is targeted at an early coding region of a gene of interest, for example, TRAC, TRBC1 and / or TRBC2, including sequence immediately after a transcription start site. within a first exon of the coding sequence, or within 500 bp of the transcription start site (for example, less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp), or within 500 bp of the start codon (for example, less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp).

[00211] In some embodiments, the target site is within an exon of the endogenous TRAC locus. In certain embodiments, the target site is within an intron of the endogenous TRAC locus. In some aspects, the target site is within the control or regulatory element, for example, a promoter, 5 'untranslated region (UTR) or 3' UTR, of the TRAC locus. In certain embodiments, the target site is within an open reading frame of an endogenous TRAC locus. In particular modalities, the target site is within an exon within the open reading structure of the TRAC locus.

[00212] In particular modalities, genetic disruption, for example, DNA breakdown, is targeted at or within an open reading frame of a gene or locus of interest, for example, TRAC, TRBC1, and / or TRBC2. In some embodiments, the genetic disruption is targeted at or within an intron within the open reading frame of a gene or locus of interest. In some modalities

However, the genetic disruption is targeted within an exon within the open reading structure of the gene or locus of interest.

[00213] In particular modalities, genetic disruption, for example, DNA breakdown, is targeted at or within an intron. In certain embodiments, genetic disruption, for example, DNA breakdown, is targeted at or within an exon. In some embodiments, the genetic disruption, for example, DNA breakdown, is targeted at or within an exon of a gene of interest, for example, TRAC, TRBC1 and / or TRBC2.

[00214] In particular modalities, genetic disruption, for example, DNA breakdown, is targeted within an exon of a TRBC gene, open reading frame, or locus, for example, TRBC1 and / or TRBC2. In certain modalities, the genetic disruption is within the first exon, second exon, third exon, or fourth exon of the TRBC1 and / or TRBC2 gene, open reading structure, or locus. In some ways, the genetic disruption is within the first exon of the TRBC1 and / or TRBC2 gene, open reading structure, or locus. In certain modalities, the genetic rupture is within the first exon, second exon, third exon, or fourth exon of the TRBC1 and / or TRBC2 gene, open reading structure or locus. In some embodiments, the genetic disruption is between most of the 5 'nucleotide of exon 1 and upstream of most of the 3' nucleotide of exon 1. In particular embodiments, the genetic disruption is within the first exon of the TRBC gene, open reading frame, or locus. In some embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream of the 5 'end of the first exon in a TRBC1 gene and / or TRBC2, open reading structure, or locus. In particular modalities, the genetic disruption is between 1 bp and 400 bp, between 50 and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp downstream of the 5 'end of the first exon in the TRBC1 gene and / or TRBC2,

open reading structure, or locus, each inclusive. In certain modalities, the genetic rupture is between 100 bp and 150 bp downstream of the 5 'end of the first exon in the TRBC1 gene and / or the TRBC2, open reading structure, or locus, inclusive.

[00215] In particular modalities, genetic disruption, for example, DNA breakdown, is targeted within an exon of a TRBC gene, open reading frame, or locus, for example, TRBC1 and / or TRBC2. In certain modalities, the genetic disruption is within the first exon, second exon, third exon, or fourth exon of the TRBC gene, open reading structure, or locus. In some embodiments, the genetic disruption is within the first exon of the TRBC gene, open reading frame, or locus. In certain embodiments, the genetic disruption is within the first exon, second exon, third exon, or fourth exon of the TRBC gene, open reading frame, or locus. In some modalities, the genetic rupture is between the nucleotide 5 'of exon 1 and upstream of nucleotide 3' of exon 1. In particular embodiments, the genetic rupture is within the first exon of the TRBC gene, open reading structure, or locus. In some embodiments, the genetic disruption is within 400 bp, 350 bp, 300 bp, 250 bp, 200 bp, 150 bp, 100 bp, or 50 bp downstream of the 5 'end of the first exon in a gene. TRBC, open reading structure, or locus. In particular modalities, the genetic break is between 1 bp and 400 bp, between 50 and 300 bp, between 100 bp and 200 bp, or between 100 bp and 150 bp downstream of the 5 'end of the first exon in the gene TRBC, open reading structure, or locus, each inclusive. In certain embodiments, the genetic disruption is between 100 bp and 150 bp downstream of the 5 'end of the first exon in the TRBC gene, open reading frame, or locus, inclusive.

2. Genetic Disruption Methods

[00216] Methods for generating genetic disruption, including those described in this document, may involve the use of one or more agents capable of inducing genetic disruption, such as, systems modified to induce genetic disruption, a cleavage and / or a double strand break (DSB) or an incision at a target site or target position in endogenous DNA so that repair of the break by a process born of error, such as non-homologous end junction (NHEJ) or repair using a repair pattern HDR could result in the knockout of the gene and / or the insertion of a sequence of interest (for example, exogenous nucleic acid sequences or transgene encoding a portion of a chimeric receptor) at or near the site target or position. Also provided are one or more agents capable of inducing a genetic disruption, for use in the methods provided in this document. In some respects, one or more agents can be used in combination with the standard nucleotides provided in this document, for targeted repair-mediated integration (HDR) of transgene sequences (for example, described in this document in Section IB) .

[00217] In some embodiments, one or more agents capable of inducing a genetic disruption comprise a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to a particular site or position in the genome, for example, a target site or target position. In some respects, targeted genetic disruption, for example, DNA breakdown or cleavage, of the endogenous genes encoding the TCR is achieved using a protein or nucleic acid that is coupled to or complexed with a gene editing nuclease , such as in a chimeric or fusion protein. In some embodiments, one or more agents capable of inducing genetic disruption comprise an RNA-guided nuclease or a fusion protein comprising a DNA targeting protein and a nuclease.

[00218] In some embodiments, the agent comprises several components, such as an RNA-guided nuclease, or a fusion protein that comprises a protein targeting DNA and a nuclease. In some embodiments, targeted genetic disruption is performed using a DNA targeting molecule that includes a DNA binding protein, such as one or more zinc finger protein (ZFP) or transcription activator-like effectors (TALEs) ), fused to a nuclease, such as an endonuclease. In some embodiments, targeted genetic disruption is performed using RNA-guided nucleases, such as, a nuclease system (Cas) associated with regularly clustered short palindromic nucleic acid (CRISPR) (including Cas and / or Cfp1). In some embodiments, targeted genetic disruption is performed using agents capable of inducing genetic disruption, such as targeted or sequence specific nucleases, including targeted DNA binding nucleases and gene editing nucleases, such as zinc finger (ZFN) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as a CRISPR-associated nuclease system (Cas), specifically designed to target at least one site target, sequence of a gene or a portion thereof. Exemplary ZFNs, TALEs, and TALENs are described in, for example, Lloyd et al., Frontiers in Immunology, 4 (221): 1-7 (2013).

[00219] Zinc finger proteins (ZFPs), transcription activator-like effectors (TALEs) and CRISPR system binding domains can be "modified" to bind to a predetermined nucleotide sequence, for example, by modification (changing one or more amino acids) of the recognition helix region of a naturally occurring ZFP or TALE protein. Modified DNA binding proteins (ZFPs or TALEs) are proteins that are non-naturally occurring. Rational criteria for the project include the application of substitution rules and computerized algorithms for processing information in a database that stores existing project information and connection data from ZFP and / or TALE. See, for example, United States Patent Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and United States Publication No. 20110301073.

[00220] In some embodiments, one or more agents specifically target at least one target site, for example, at or near a gene of interest, for example, TRAC, TRBC1 and / or TRBC2. In some embodiments, the agent comprises a combination of ZFN, TALEN or CRISPR / Cas9 that specifically binds to, recognizes or hybridizes to target sites. In some embodiments, the CRISPR / Cas9 system includes a modified crRNA / TRACr RNA ("single-guide RNA") to guide specific cleavage. In some modalities, the agent comprises nucleases based on the Argonaute system (for example, from T. thermophilus, known as 'TtAgo', (Swarts et al., (2014) Nature 507 (7491): 258-261) Targeted cleavage using any of the nuclease systems described in this document can be exploited to insert the sequences of a transgene, for example, nucleic acid sequences encoding a recombinant receptor, at a specific target location, for example, in endogenous TCR genes, using processes mediated by HDR or NHEJ.

[00221] In some embodiments, a "zinc finger DNA binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through of one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through the coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Among the ZFPs are artificial ZFP domains targeting specific DNA sequences, typically 9-18 nucleotides in length, generated by assembling individual fingers. ZFPs include those in which a single finger domain is approximately 30 amino acids in length and contains an alpha helix that contains two invariant histidine residues coordinated through zinc with two single-loop beta cysteines, and having two , three, four, five or six fingers. Generally, the sequence specificity of a ZFP can be altered by making amino acid substitutions in the four positions of the helix (-1, 2, 3, and 6) in a zinc finger recognition helix. Thus, for example, the ZFP or molecule that contains ZFP is non-naturally occurring, for example, it is modified to bind to a target site of choice.

[00222] In some cases, the DNA targeting molecule is or comprises a zinc finger DNA binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). For example, fusion proteins comprise the cleavage domain (or cleavage half-domain) of at least one Type IIS restriction enzyme and one or more zinc finger binding domains, which may or may not be modified. In some cases, the cleavage domain is that of the FokI Type IIS restriction endonuclease, which generally catalyzes the cleavage of double-stranded DNA into 9 nucleotides from its recognition site in one strand and 13 nucleotides from its recognition site in another. See, for example, United States Patent Nos. 5,356,802; 5,436,150 and 5,487,994; Li et al. (1992) Proc. Natl. Acad. Sci. USA 89: 4275-4279; Li et al. (1993) Proc. Natl. Acad. Sci. USA 90: 2764-2768; Kim et al. (1994a) Proc. Natl.

Acad. Sci. USA 91: 883-887; Kim et al. (1994b) J. Biol. Chem. 269: 978-982. Some gene-modified modified zinc fingers are commercially available. For example, a platform called CompoZr is available for zinc finger construction that specifically supplies targeted zinc fingers to thousands of targets. See, for example, Gaj et al., Trends in Biotechnology, 2013, 31 (7), 397-405. In some cases, commercially available zinc fingers are used or customized.

[00223] In some modalities, one or more target sites, for example, within TRAC, TRBC1 and / or TRBC2 genes can be targeted for genetic disruption by modified ZFNs. Exemplary ZFNs that target endogenous T cell receptor (TCR) genes include those described in, for example, US 2015/0164954, US 2011/0158957, US 2015/0056705, US 8956828 and Torikawa et al. (2012) Blood 119: 5697-5705, whose descriptions are incorporated by reference in their entirety, or those set out in any of SEQ ID NOS: 213-224 (TRAC) or SEQ ID NOS: 225 and 226 (TRBC).

[00224] Transcription activator type effector (TALE) are proteins of the bacterial species Xanthomonas that comprise a plurality of repeated sequences, each repetition comprising di-residues in position 12 and 13 (RVD) that are specific to each nucleotide base of the targeted nucleic acid sequence. Binding domains with modular base-to-base nucleic acid (MBBBD) binding properties can also be derived from different bacterial species. The new modular proteins have the advantage of showing more sequence variability than TAL repetitions. In some modalities, RVDs associated with the recognition of different nucleotides are HD for recognition of C, NG for recognition of T, NI for recognition of A, NN for recognition

G or A, NS for recognition of A, C, G or T, HG for recognition of T, IG for recognition of T, NK for recognition of G, HA for recognition of C, ND for recognition of C, HI for recognition of C, HN for recognition of G, NA for recognition of G, SN for recognition of G or A and YG for recognition of T, TL for recognition of A, VT for recognition of A or G and SW for recognition of A. In some modalities, critical amino acids 12 and 13 can be mutated towards other amino acid residues in order to modulate their specificity for nucleotides A, T, C and G and in particular to enhance this specificity.

[00225] In some embodiments, a "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat units / domains. The repeat domains, each comprising a variable repeat residue (RVD), are involved in binding TALE to its cognate target DNA sequence. A single "repeat unit" (also called a "repeat") is usually 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. TALE proteins can be designed to bind to a target site using canonical or non-canonical RVDs within the repeating units. See, for example, United States Patent Nos.

8,586,526 and 9,458,205.

[00226] In some embodiments, a "TALE nuclease" (TALEN) is a fusion protein comprising a nucleic acid binding domain typically derived from a transcription-activating effector (TALE) and a nuclease catalytic domain that cleaves a target nucleic acid sequence. The catalytic domain comprises a nuclease domain or a domain with endonuclease activity,

such as, for example, I-TevI, ColE7, NucA and Fok-I. In a particular embodiment, the TALE domain can be fused to a meganuclease such as, for example, I-CreI and I-OnuI or functional variant thereof. In some modalities, TALEN is a monomeric TALEN. A monomeric TA-LEN is a TALEN that does not require dimerization for specific recognition and cleavage, such as fusions of modified TAL repeats with the catalytic domain of I-TevI as described in WO

2012138927. TALENs have been described and used for targeting genes and gene modifications (see, for example, Boch et al. (2009) Science 326 (5959): 1509-12.; Moscow and Bogdanove (2009) Science 326 (5959): 1501; Christian et al. (2010) Genetics 186 (2): 757-61; Li et al. (2011) Nucleic Acids Res 39 (1): 359-72).

[00227] In some modalities, the TRAC, TRBC1 and / or TRBC2 genes can be targeted for genetic disruption by modified TALENs. The exemplary TALEN that targets endogenous T cell receptor (TCR) genes includes those described in, for example, WO 2017/070429, WO 2015/136001, US20170016025 and US20150203817, the descriptions of which are incorporated by reference in their entirety .

[00228] In some modalities, a "TtAgo" is an Argonaut prokaryotic protein that is believed to be involved in gene silencing. TtAgo is derived from the bacterium Thermus thermophilus. See, for example, Swarts et al (2014) Nature 507 (7491): 258-261; G. Sheng et al., (2013) Proc. Natl. Acad. Sci. U.S.A. 111, 652. A "TtAgo system" is all the necessary components, including, for example, guide DNAs for cleavage by a TtAgo enzyme.

[00229] In some modalities, a modified zinc finger protein, TALE protein or CRISPR / Cas system is not found in nature and whose production results mainly from an empirical process such as phage display, interaction trap or hybrid selection. See, for example, U.S. Patent No. 5,789,538; United States Patent No. 5,925,523; U.S. Patent No.

6,007,988; United States Patent No. 6,013,453; United States Patent No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970; WO 01/88197 and WO 02/099084.

[00230] The zinc finger and TALE DNA binding domains can be modified to bind to a predetermined nucleotide sequence, for example, by modifying (changing one or more amino acids) of the helix recognition region a naturally occurring zinc finger protein or by modifying the amino acids involved in DNA binding (the variable repeat residue or RVD region). Therefore, modified zinc finger proteins or TALE proteins are proteins that are non-naturally occurring. Non-limiting examples of methods for modifying zinc finger proteins and TALEs are design and selection. A designated protein is a protein that does not occur in nature, whose design / composition results mainly from rational criteria. Rational criteria for the project include the application of substitution rules and computerized algorithms for processing information in a database that stores information about existing ZFP or TALE projects (canonical and non-canonical RVDs) and alloy data - dog. See, for example, United States Patent Nos. 9,458,205;

8,586,526; 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.

[00231] Various methods and compositions for targeted genomic DNA cleavage have been described. These targeted cleavage events can be used, for example, to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences and facilitate targeted recombination at a predetermined chromosomal locus. See, for example, United States Patent Nos. 9,255,250;

9,200,266; 9,045,763; 9,005,973; 9,150,847; 8,956,828; 8,945,868;

8,703,489; 8,586,526; 6,534,261; 6,599,692; 6,503,717; 6,689,558;

7,067,317; 7,262,054; 7,888,121; 7,972,854; 7,914,796; 7,951,925;

8,110,379; 8,409,861; US Patent Publication 20030232410; 20050208489; 20050026157; 20050064474; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983; 20130196373; 20140120622; 20150056705; 20150335708; 20160030477 and 20160024474, whose descriptions are incorporated by reference in their entirety. Also provided are one or more agents capable of introducing a genetic disruption. Also provided are polynucleotides (for example, nucleic acid molecules) that encode one or more components of one or more agents capable of inducing a genetic disruption. The. Crispr / Cas9

[00232] In some embodiments, targeted genetic disruption, for example, DNA breakdown, of endogenous genes encoding TCR, such as TRAC and TRBC1 or TRBC2 in humans, is performed using regularly spaced short palindromic repeats grouped (CRISPR) and CRISPR-associated proteins (Cas). See, Sander and Joung, (2014) Nature Biotechnology, 32 (4): 347-355.

[00233] In general, "CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of genes associated with CRISPR ("Cas"), including sequences encoding a Cas gene, a sequence of TRACr (CRISPR transactivator) (for example, TRACrRNA or an active partial TRACrRNA), a TRACr-mate sequence (comprising a "direct repeat" and a partial direct repeat processed by the

TRACrRNA in the context of an endogenous CRISPR system), a guide sequence (also referred to as a "spacer" in the context of an endogenous CRISPR system), and / or other sequences and transcriptions of a CRISPR locus.

[00234] In some respects, the CRISPR / Cas nuclease system or CRISPR / Cas nuclease includes a non-coding guide RNA (gRNA), the sequence of which specifically binds to DNA, and a Cas protein (for example, Cas9), with nuclease functionality. 1) Guide RNA (gRNA)

[00235] In some embodiments, one or more agents comprise at least one of: a guide RNA (gRNA) having a targeting domain that is complementary with a target site of a TRAC gene; a gRNA having a targeting domain that is complementary to a target site of one or both of the TRBC1 and TRBC2 genes; or at least one nucleic acid that encodes a gRNA.

[00236] In some respects, a "gRNA molecule" is for a nucleic acid that promotes the specific targeting or homing of a complex of gRNA molecule / Cas9 molecule to a target nucleic acid, such as a locus in genomic DNA of a cell. The gRNA molecules can be unimolecular (having a single RNA molecule), sometimes referred to herein as "chimeric" gRNAs, or modular (comprising more than one, and usually two, separate RNA molecules). In general, a guide sequence, for example, guide RNA, is any polynucleotide sequence that comprises at least a portion of the sequence that has sufficient complementarity with a target polynucleotide sequence, such as the TRAC, TRBC1 and / or TRBC2 in humans, to hybridize to the target sequence at the target site and direct sequence specific binding of the CRISPR complex to the target sequence. In some modalities, in the context of the formation of a

CRISPR complex, "target sequence" generally refers to a sequence for which a guide sequence is designed to be complementary, where hybridization between the target sequence and the domain, for example, target domain, of the guide RNA promotes the formation of a CRISPR complex. Full complementarity is not necessarily necessary, as long as there is sufficient complementarity to cause hybridization and promote the formation of a CRISPR complex. Generally, a guide sequence is selected to reduce the degree of secondary structure within the guide sequence. The secondary structure can be determined by any suitable polynucleotide folding algorithm.

[00237] In some embodiments, a guide RNA (gRNA) specific to a locus of target interest (for example, in the TRAC, TRBC1 and / or TRBC2 in humans) is used for RNA-guided nuclease, for example, Cas, for induce a DNA break at the target site or target position. Methods for designating exemplary gRNAs and targeting domains may include those described in, for example, International PCT Publication Nos. WO 2015/161276, WO 2017/193107 and WO 2017/093969 US2016 / 272999 and US2015 /

056705.

[00238] Several examples of gRNA structures, with indicated domains, are described in WO 2015/161276, for example, in FIGS. 1A-1G on it. Although not wishing to be limited by theory, with regard to the three-dimensional shape, or intra- or inter-tape interactions of an active form of a gRNA, regions of high complementarity are sometimes shown as duplexes in WO 2015/161276, for example, in FIGS. 1A-1G on it and other representations provided in this document.

[00239] In some cases, gRNA is a unimolecular or chimeric gRNA that comprises, from 5 'to 3': a targeting domain that targets a target site or position, such as within a sequence of the TRAC locus (exemplary nucleotide sequence the human TRAC gene locus in SEQ ID NO: 1; NCBI reference sequence: NG_001332.3, TRAC; exemplary genomic sequence described in Table 1 of this document); a first domain of complementarity; a link domain; a second complementarity domain (which is complementary to the first complementarity domain); a proximal domain; and optionally, a tail domain. In some cases, the gRNA is a unimolecular or chimeric gRNA that comprises, from 5 'to 3': a targeting domain that targets a target site or position, such as within a sequence of TRBC1 or TRBC2 locus (exemplary nucleotide sequence of the human TRBC1 gene locus established in SEQ ID NO: 2; NCBI reference sequence: NG_001333.2, TRBC1; exemplary genomic sequence described in Table 2 herein; exemplary nucleotide sequence of the locus of human TRBC2 gene established in SEQ ID NO: 3; NCBI reference sequence: NG_001333.2, TRBC2; exemplary genetic sequence described in Table 3 in this document); a first domain of complementarity; a link domain; a second domain of complementarity (which is complementary to the first domain of complementarity); a proximal domain; and optionally, a tail domain.

[00240] In other cases, gRNA is a modular gRNA comprising the first and the second strands. In these cases, the first strand preferably includes, from 5 'to 3': a targeting domain (which targets a target site or position, such as, within a sequence of the TRAC locus (exemplary nucleotide sequence of the gene locus) Human TRAC established in SEQ ID NO: 1; NCBI reference sequence: NG_001332.3, TRAC; exemplary genomic sequence described in Table 1 in this document) or TRBC1 or TRBC2 locus (se-

exemplary nucleotide sequence of the human TRBC1 gene locus set forth in SEQ ID NO: 2; NCBI reference sequence: NG_001333.2, TRBC11; exemplary genomic sequence described in Table 2 in this document; exemplary nucleotide sequence of the human TRBC2 gene locus set forth in SEQ ID NO: 3; NCBI reference sequence: NG_001333.2, TRBC2); and a first domain of complementarity. The second tape generally includes, from 5 'to 3': optionally, a 5 'extension domain; a second domain of complementarity; a proximal domain; and optionally, a tail domain. A) Targeting domain

[00241] Examples of placement of targeting domains include those described in WO 2015/161276, for example, in FIGS. 1A-1G on it. The targeting domain comprises a nucleotide sequence that is complementary, for example, at least 80, 85, 90, 95, 98 or 99% complementary, for example, completely complementary, to the target sequence in the target nucleic acid. The target nucleic acid strand comprising the target sequence is referred to herein as the "complementary strand" of the target nucleic acid. Guidance on selecting targeting domains can be found, for example, in Fu Y et al., Nat Biotechnol 2014 (doi: 10.1038 / nbt.2808) and Sternberg SH et al., Nature 2014 (doi : 10.1038 / natural13011).

[00242] The targeting domain is part of an RNA molecule and will therefore comprise the uracil base (U), while any DNA encoding the gRNA molecule will comprise the thymine base (T). Although not wishing to be limited to theory, in some modalities, it is believed that the complementarity of the targeting domain with the target sequence contributes to the specificity of the interaction of the gRNA / Cas9 molecule complex with an acid target nucleic acid. It is understood that in a target sequence pair and targeting domain, the uracil bases in the targeting domain will pair with the adenine bases in the target sequence. In some modalities, the target domain itself comprises in the 5 'to 3' direction, an optional secondary domain and a core domain. In some embodiments, the core domain is completely complementary to the target sequence. In some embodiments, the targeting domain is 5 to 50 nucleotides in length. The target nucleic acid strand with which the targeting domain is complementary is referred to herein as the complementary strand. Some or all of the nucleotides in the domain may have a modification, for example, to make it less susceptible to degradation, to improve biocompatibility, etc. By way of non-limiting example, the main chain of the target domain can be modified with a phosphorothioate, or other modification (s). In some cases, a targeting domain nucleotide may comprise a 2 'modification, for example, a 2-acetylation, for example, a 2' methylation or other modification (s).

[00243] In several modalities, the targeting domain is 16–26 nucleotides in length (ie, it is 16 nucleocides in length, or 17 nucleotides in length, or 18, 19, 20, 21, 22, 23, 24 , 25 or 26 nucleotides in length B) Exemplary Targeting Domains

[00244] Exemplary targeting domains contained within the gRNA to target the genetic disruption of human TRAC, TRBC1 or TRBC2 include those described in, for example, WO 2015/161276, WO 2017/193107, WO 2017/093969, US2016 / 272999 and US2015 / 056705 or a targeting domain that can bind to the targeting sequences described previously. Exemplary targeting domains contained within the gRNA for

viewing the genetic disruption of the human TRAC locus using Cas9 of S. pyogenes or S. aureus can include any of those set out in Table -4. Table 4. Exemplary TRAC gRNA targeting domain sequences Targeting domain name SEQ gRNA species Cas9 ID NO: TRAC-10 UCUCUCAGCUGGUACACGGC S. pyogenes 28 TRAC-110 UGGAUUUAGAGUCUCUCAGC S. pyogenes 29 TRAC-116 ACACGGUAG TRAC-16 GAGAAUCAAAAUCGGUGAAU S. pyogenes 31 TRAC-4 GCUGGUACACGGCAGGGUCA S. pyogenes 32 TRAC-49 CUCAGCUGGUACACGGC S. pyogenes 33 TRAC-2 UGGUACACGGCAGGGUC S. pyogenes 43 TRAC-AGGU UCAGCUGGUACACGGCA S. pyogenes 23 37 34 TRAC-TRAC 38 GCAGACAGACUUGUCAC S. pyogenes S. pyogenes GGUACACGGCAGGGUCA-25 39 128 TRAC-TRAC 40 CUUCAAGAGCAACAGUGCUG S. pyogenes S. pyogenes AGAGCAACAGUGCUGUGGCC-105 41 106-TRAC AAAGUCAGAUUUGUUGCUCC 42 S. pyogenes 123 Trac-ACAAAACUGUGCUAGACAUG S. pyogenes 43 TRAC-64 AAACUGUGCUAGACAUG S. pyogenes 44 TRAC-97 UGUGCUAGACAUGAGGUCUA S. pyogenes 45 TRAC-148 GGCUGGGGAAGAAGGUGUCUUC S. aureus 46 TRAC-147 GCUGGGGAAGAAGGUGUCUUC S. aureu s 47 TRAC-234 GGGGAAGAAGGUGUCUUC S. aureus 48 TRAC-167 GUUUUGUCUGUGAUAUACACAU S. aureus 49 TRAC-177 GGCAGACAGACUUGUCACUGGAU S. aureus 50

U TRAC-176 GCAGACAGACUUGUCACUGGAUU S. aureus 51 TRAC-257 GACAGACUUGUCACUGGAUU S. aureus 52

Targeting domain name SEQ gRNA species Cas9 ID NO: TRAC-233 GUGAAUAGGCAGACAGACUUGUC S. aureus 53

TRAC-231 GAAUAGGCAGACAGACUUGUCA S. aureus 54 TRAC-163 GAGUCUCUCAGCUGGUACACGG S. aureus 55 TRAC-241 GUCUCUCAGCUGGUACACGG S. aureus 56 TRAC-179 GGUACACGGCAGGGUCAGGGUU S. aurek 57

[00245] Exemplary targeting domains contained within the gRNA to target the genetic disruption of the human TRBC1 or TRBC2 locus using S. pyogenes or S. aureus Cas9 can include any of those set out in Table 5 Table 5. Targeting domain sequences specimen TRBC1 or TRBC2 gRNA targeting domain name SEQ species gRNA Cas9 ID NO: TRBC-40 CACCCAGAUCGUCAGCGCCG S. pyogenes 59 TRBC-52 CAAACACAGCGACCUCGGGU S. pyogenes 60 TRBC-25 UGACGAGUGGACUCAGAGGAGGACGA pyogenes 62 TRBC-50 GGCCUCGGCGCUGACGAUCU S. pyogenes 63 TRBC-39 GAAAAACGUGUUCCCACCCG S. pyogenes 64 TRBC-49 AUGACGAGUGGACCCAGGAU S. pyogenes 65 TRBC-51 AGUCCAGUUCUACGGGCUCU S. pyogenes 66 TRBC-AGAGGGUCCCAGGU TRBC-45 UCAAACACAGCGACCUCGGG S. pyogenes 69 TRBC-34 CGUAGAACUGGACUUGACAG S. pyogenes 70 TRBC-227 AGGCCUCGGCGCUGACGAUC S. pyogenes 71 TRBC-41 UGACAGCGGAAGUGGUUGCG S. pyog enes 72 TRBC-30 UUGACAGCGGAAGUGGUUGC S. pyogenes 73 TRBC-206 UCUCCGAGAGCCCGUAGAAC S. pyogenes 74 Targeting domain name SEQ gRNA species Cas9 ID NO: TRBC-32 CGGGUGGGAACACGUUUUUC S. pyogenes 76 TRBC-GACU 276 S. pyogenes GAUCGUCAGCGCCGAGGCCU 77 TRBC-230 S. pyogenes GGCUCAAACACAGCGACCUC 78 TRBC-235 S. pyogenes UGAGGGUCUCGGCCACCUUC-TRBC 79 38 80 TRBC AGGCUUCUACCCCGACCACG S. pyogenes S. pyogenes CCGACCACGUGGAGCUGAGC-223 81 221-TRBC UGACAGGUUUGGCCCUAUCC 82 TRBC S. pyogenes S-48 CUUGACAGCGGAAGUGGUUG pyogenes 83 TRBC-216 AGAUCGUCAGCGCCGAGGCC S. pyogenes 84 TRBC-210 GCGCUGACGAUCUGGGUGAC S. pyogenes 85 TRBC-268 UGAGGGCGGGCUGCUCCUUG S. pyogenes 86 TRBC-193 GUUGCGGGGGUGGACCAGGGGGUCUCCAGGGGGUCGGGGGGUCGGGGGGGUCGGGGGGGGGGGGGUCGGGGGGGGGGGGGGGUCGG 89 TRBC-43 GCGGGGGUUCUGCCAGAAGG S. pyogenes 90 TRBC-272 UGGCUCAAACACAGCGACCU S. pyogenes 91 TRBC-33 ACUGGACUUGACAGCGGAAG S. pyogenes 92 TRBC-44 GACAGCGGAAGUGGUUGCGG S. pyogenes 93 TRBC-211 GCUGUCAAGUCCAGUUCUAC S. pyogenes 94 TRBC-253 GUAUCUGGAGUCAUUGAGGG S. pyogenes 95 TRBC-18 CUCGGCGCUGACGAUCU S. pyogenes 96 TRBC-6 CCUCGGCGCUGACGAUC S. pyogenes 97 TRBC-85 CCGGAGAGGAC 98G TRBC-93 GAAUGACGAGUGGACCC S. pyogenes 100 TRBC-415 GGGUGACAGGUUUGGCCCUAUC S. aureus 101 TRBC-414 GGUGACAGGUUUGGCCCUAUC S. aureus 102 TRBC-310 GUGACAGGUUUGGCCCUAUC S. aureus 103 TRBC-30UGGAGGCCCCCCUAC Targeting domain SEQ gRNA species Cas9 ID NO: TRBC-468 GACCACGUGGAGCUGAGCUGGU S. aureus 106

GG TRBC-462 GUGGAGCUGAGCUGGUGG S. aureus 107 TRBC-424 GGGCGGGCUGCUCCUUGAGGGG S. aureus 108

CU TRBC-423 GGCGGGCUGCUCCUUGAGGGGC S. aureus 109

U TRBC-422 GCGGGCUGCUCCUUGAGGGGCU S. aureus 110 TRBC-420 GGGCUGCUCCUUGAGGGGCU S. aureus 111 TRBC-419 GGCUGCUCCUUGAGGGGCU S. aureus 112 TRBC-418 GCUGCUCCUUGAGGGGA SCAGGGA

G TRBC-444 GUGAAUGGGAAGGAGGUGCACAG S. aureus 115 TRBC-442 GAAUGGGAAGGAGGUGCACAG S. aureus 116

[00246] In some embodiments, the gRNA for targeting TRAC, TRBC1 and / or TRBC2 can be described in this document or described elsewhere, for example, in WO 2015/161276, WO 2017/193107, WO 2017/093969, US2016 / 272999 and US2015 / 056705 or a targeting domain that can be linked to the targeting sequences described above. In some embodiments, the sequence targeted by the CRISPR / Cas9 gRNA at the TRAC gene locus is set out in SEQ ID NOS: 117, 163 and 165-211, such as, GAGAATCAAAATCGGTGAAT (SEQ ID NO: 163) or ATTCACCGATTTTGATTCTC (SEQ ID NO : 117). In some embodiments, the sequence targeted by the CRISPR / Cas9 gRNA at the TRBC1 and / or TRBC2 gene locus is set out in SEQ ID NOS: 118, 164 and 212, such as, GGCCTCGGCGCTGA- CGATCT (SEQ ID NO: 164) or GATCGTCAGCGCCGAGGCC (SEQ ID NO: 118). In some embodiments, the gRNA targeting domain sequence to target a target site at the TRAC gene locus is GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31). In some embodiments, the gRNA targeting domain sequence to target a target site at the TRBC1 and / or TRBC2 gene locus is GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63).

[00247] In some embodiments, the gRNA for directing the TRAC gene locus can be obtained by in vitro transcription if- quence AGCGCTCTCGTACAGAGTTGGCATTATAATACGACTCAC- TATAGGGGAGAATCAAAATCGGTGAATGTTTTAGAGCTAGAAA- TAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAG- TGGCACCGAGTCGGTGCTTTTTTT (set forth in SEQ ID NO: 26; bold and underlined portion is complementary to the target site at the TRAC locus), or chemically synthesized, where the gRNA had the sequence 5'- GAG AAU CAA AAU CGG UGA AUG UUU UAG AGC UAG AAA

UAG CAA GUU AAA AUA AGG CUA GUC CGU UAU CAA CUU GAA AAA GUG GCA CCG AGU CGG UGC UUU U -3 '(established in SEQ ID NO: 27; see, Osborn et al., Mol Ther. 24 (3): 570 -581 (2016)). Other exemplary gRNA sequences for generating the genetic disruption of endogenous genes encoding TCR domains or regions, for example, TRAC, TRBC1 and / or TRBC2 are described, for example, in International PCT Publication No. WO 2015/161276. Exemplary methods for editing genes from the endogenous TCR locus include those described in, for example, United States Publications Nos. US2011 / 0158957, US2014 / 0301990, US2015 / 0098954, US2016 / 0208243; US2016 / 272999 and US2015 / 056705; International PCT Publication Nos. WO 2014/191128, WO 2015/136001, WO 2015/161276, WO 2016/069283, WO 2016/016341; and Osborn et al. (2016) Mol. Ther. 24 (3): 570-581. Any of the known methods can be used to generate a genetic disruption of the endogenous genes that encode TCR domains or regions can be used in the modalities provided in this document.

[00248] In some embodiments, targeting domains include those for introducing a genetic disruption at the TRAC, TRBC1 and / or TRBC2 locus using Cas9 from S. pyogenes or using Cas9 from N. meningitidis.

[00249] In some embodiments, targeting domains include those for introducing a genetic disruption at the TRAC, TRBC1 and / or TRBC2 locus using Cas9 from S. pyogenes. Any of the targeting domains can be used with a Cas9 molecule from S. pyogenes that generates a double strand break (Cas9 nuclease) or a single strand break (nickase cas9).

[00250] In some modalities, double targeting is used to create two nicknames on opposite strands of DNA using Cas9 S. pyogenes nickases with two targeting domains that are complementary to the opposite strands of DNA, for example, a gRNA that com- any negative tape targeting domain can be paired with any gRNA comprising a plus tape targeting domain. In some embodiments, the two gRNAs are oriented in the DNA so that the PAMs are facing outward and the distance between the 5 'ends of the gRNAs is 0-50bp. In some embodiments, two gRNAs are used to target two Cas9 nucleases or two Cas9 nickases, for example, using a Cas9 molecule / gRNA molecule complex pair guided by two different gRNA molecules to cleave the target domain with two single-strand breaks on opposite strands of the target domain. In some modalities, the two Cas9 nickases may include a molecule with HNH activity, for example, a Cas9 molecule having inactivated RuvC activity, for example, a Cas9 molecule having a D10 mutation, for example, the D10A mutation, a molecule having RuvC activity, for example, a Cas9 molecule having inactivated HNH activity, for example, a Cas9 molecule having a mutation in H840, for example, an H840A, or a molecule having RuvC activity, for example, a molecule Cas9 having HNH activity inactivated, for example, a Cas9 molecule with a mutation in N863, for example, N863A. In some embodiments, each of the two gRNAs is complexed with a Cas9 D10A nickase.

[00251] In some embodiments, the target sequence (target domain) is at or near the TRAC, TRBC1 and / or TRBC2 locus, such as any part of the TRAC, TRBC1 and / or TRBC2 coding sequence set out in SEQ ID NO: 1-3 or described in Tables 1-3 in this document. In some embodiments, the target nucleic acid complementary to the targeting domain is located in an early coding region of a gene of interest, such as, TRAC, TRBC1 and / or TRBC2. Targeting the early coding region can be used to genetically disrupt (ie, eliminate expression of) the gene of interest. In some embodiments, the early coding region of a gene of interest includes sequence immediately after a start codon (eg, ATG), or within 500 bp of the start codon (eg, less than 500, 450 , 400, 350, 300, 250, 200, 150, 100, 50 bp, 40 bp, 30 bp, 20 bp, or 10 bp). In particular examples, the target nucleic acid is within 200 bp, 150 bp, 100 bp, 50 bp, 40 bp, 30 bp, 20 bp or 10 bp of the start codon. In some examples, the gRNA targeting domain is complementary, for example, at least 80, 85, 90, 95, 98 or 99% complementary, for example, completely complementary to the target sequence in the target nucleic acid, such as, the target nucleic acid at the TRAC, TRBC1 and / or TRBC2 locus.

[00252] In some respects, the gRNA may target a site within an exon of the open reading frame of the endogenous locus TRAC, TRBC1 and / or TRBC2. In some respects, the gRNA may target a site within an intron of the open reading frame of the TRAC, TRBC1 and / or TRBC2 locus. In some respects, the gRNA may target a site within a control or regulatory element, for example, a promoter, from the TRAC, TRBC1 and / or TRBC2 locus. In some respects, the target site at the TRAC, TRBC1 and / or TRBC2 locus that is targeted by the gRNA can be any target site described in this document, for example, in Section I.A.1. In some embodiments, the gRNA may target a site within or near exons corresponding to the early coding region, for example, exon 1, 2 or 3 of the open reading structure of the endogenous site TRAC, TRBC1 and / or TRBC2, or including sequence immediately after a transcription initiation site, within exon 1, 2, or 3, or within less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 1, 2, or 3. In some embodiments, the gRNA may target a site at or near exon 2 of the endogenous site TRAC, TRBC1 and / or TRBC2, or within less than 500 , 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp of exon 2. C) First domain of complementarity

[00253] Examples of first domains of complementarity include those described in WO 2015/161276, for example, in FIGS. 1A-1G on it. The first domain of complementarity is complementary to the second domain of complementarity described in this document, and generally has sufficient complementarity for the second domain of complementarity to form a duplexed region under at least some physiological conditions. The first complementarity domain is typically 5 to 30 nucleotides in length, and can be 5 to 25 nucleotides in length, 7 to 25 nucleotides in length, 7 to 22 nucleotides in length, 7 to 18 nucleotides in length, or 7 to 15 nucleotides in length. In several modalities, the first domain of com-

Plementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length.

[00254] Normally, the first complementarity domain does not have exact complementarity with the second complementarity domain target. In some embodiments, the first complementarity domain may have 1, 2, 3, 4 or 5 nucleotides that are not complementary to the corresponding nucleotide in the second complementarity domain. For example, a segment of 1, 2, 3, 4, 5 or 6, (for example, 3) nucleotides from the first complementary domain may not match in the duplex, and may form a non-duplexed or skipped region. In some cases, a broken or skipped region, for example, a 3 nucleotide jump, is present in the second domain of complementarity. This unpaired region optionally begins with 1, 2, 3, 4, 5, or 6, for example 4, nucleotides from the 5 'end of the second complementarity domain.

[00255] The first complementarity domain can include 3 subdomains, which, in the 5 'to 3' direction are: a 5 'subdomain, a central subdomain and a 3' subdomain. In some embodiments, the subdomain 5 'is 4-9, for example, 4, 5, 6, 7, 8 or 9 nucleotides in length. In some embodiments, the central subdomain is 1, 2, or 3, for example, 1, nucleotide in length. In some modes, subdomain 3 'is 3 to 25, for example, 4-22, 4-18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25, nucleotides in length.

[00256] In some modalities, the first and second complementarity domains, when duplexed, comprise 11 paired nucleotides, for example, in the sequence of gRNA (an underlined stripe, one in bold):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAUAGCAAG

UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCG AGUCGGUGC (SEQ ID NO: 144).

[00257] In some modalities, the first and second complementary domains, when duplexed, comprise 15 paired nucleotides, for example, in the sequence of gRNA (an underlined stripe, one in bold):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGAAAAGC

AUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAG UGGCACCGAGUCGGUGC (SEQ ID NO: 145).

[00258] In some modalities the first and second complementarity domains, when duplexed, comprise 16 paired nucleotides, for example, in the sequence of gRNA (an underlined ribbon, one in bold):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGGAAACA

GCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAA AGUGGCACCGAGUCGGUGC (SEQ ID NO: 146).

[00259] In some modalities the first and second complementarity domains, when duplexed, comprise 21 paired nucleotides, for example, in the sequence of gRNA (an underlined stripe, one in bold):

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAUGCUGUUUUGG

AAACAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAU CAACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 147).

[00260] In some embodiments, nucleotides are exchanged to remove poly-U TRACts, for example, in the sequence of gRNAs (underlined nucleotide exchanges):

NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAGAAAUAGCAAGU

UAAUAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGC (SEQ ID NO: 148);

NNNNNNNNNNNNNNNNNNNNGUUUAAGAGCUAGAAAUAGCAAGU

UUAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGA GUCGGUGC (SEQ ID NO: 149); and

NNNNNNNNNNNNNNNNNNNNGUAUUAGAGCUAUGCUGUAUUGG

AAACAAUACAGCAUAGCAAGUUAAUAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 150).

[00261] The first complementarity domain may share homology with, or be derived from, a naturally occurring first complementary domain. In some embodiments, it has at least 50% homology with a first domain of complementarity in this document described, for example, a first domain of complementarity S. pyogenes, S. aureus, N. mening- tidis, or S thermophilus.

[00262] It should be noted that one or more, or even all nucleotides in the first complementarity domain, may have a change along the lines discussed in this document for the targeting domain. D) The connection domain

[00263] Examples of binding domains include those described in WO 2015/161276, for example, in FIGS. 1A-1G on it. In a unimolecular or chimeric gRNA, the binding domain serves to link the first complementarity domain with the second complementarity domain of a unimolecular gRNA. The linker domain can link the first and second complementarity domains covalently or non-covalently. In some embodiments, the bond is covalent. In some embodiments, the binding domain covalently couples the first and second complementary domains, see, for example, WO 2015/161276, for example, in FIGS. 1B-1E in it. In some embodiments, the bonding domain is, or comprises, a covalent bond interposed between the first complementarity domain and the second complementary domain.

tarity. Usually the binding domain comprises one or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, but in various embodiments the linker can be 20, 30, 40, 50 or even 100 nucleotides in length.

[00264] In modular gRNA molecules, the two molecules are associated due to the hybridization of the complementary domains and a binding domain may not be present. See, for example, WO 2015/161276, for example, in FIG. 1A in it.

[00265] A wide variety of binding domains are suitable for use in unimolecular gRNA molecules. Binding domains may consist of a covalent bond, or be as short as one or a few nucleotides, for example, 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, a binding domain is 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 or more nucleotides in length. In some embodiments, a binding domain is 2 to 50, 2 to 40, 2 to 30, 2 to 20, 2 to 10, or 2 to 5 nucleotides in length. In some embodiments, a binding domain shares homology with, or is derived from, a naturally occurring sequence, for example, the sequence of a TRACrRNA that is 5 'for the second complementarity domain. In some embodiments, the binding domain has at least 50% homology with a binding domain in the present document described.

[00266] As discussed in this document in connection with the first domain of complementarity, some or all of the nucleotides of the binding domain may include a modification. E) 5 'Extension Domain

[00267] In some cases, a modular gRNA may comprise the additional sequence, 5 'for the second complementarity domain, in the present document referred to as the 5' extension domain, WO 2015/161276, for example, in FIG. 1A in it. In some modalities

the 5 'extension domain is 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, or 2-4 nucleotides in length. In some embodiments, the 5 'extension domain is 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides in length. F) Second Complementarity Domain

[00268] Examples of second complementarity domains include those described in WO 2015/161276, for example, in FIGS. 1A-1G on it. The second domain of complementarity is complementary to the first domain of complementarity, and generally has sufficient complementarity for the second domain of complementarity to form a duplexed region under at least some physiological conditions. In some cases, for example, as shown in WO 2015/161276, for example, in FIG. 1A-1B therein, the second complementarity domain may include a sequence that lacks complementarity with the first complementarity domain, for example, a sequence that exits the duplexed region.

[00269] The second complementarity domain can be 5 to 27 nucleotides in length, and in some cases it can be longer than the first complementarity region. For example, the second domain of complementarity can be 7 to 27 nucleotides in length, 7 to 25 nucleotides in length, 7 to 20 nucleotides in length, or 7 to 17 nucleotides in length. More generally, the complementarity domain can be 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , or 26 nucleotides in length.

[00270] In some modalities, the second domain of complementarity comprises 3 subdomains, which, in the 5 'to 3' direction are: a 5 'subdomain, a central subdomain and a 3' subdomain. In some embodiments, subdomain 5 'is 3 to 25, for example, 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,

17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the central subdomain is 1, 2, 3, 4 or 5, for example, 3, nucleotides in length. In some embodiments, subdomain 3 'is 4 to 9, for example, 4, 5, 6, 7, 8 or 9 nucleotides in length.

[00271] In some embodiments, subdomain 5 'and subdomain 3' of the first domain of complementarity are respectively complementary, for example, fully complementary, with subdomain 3 'and subdomain 5' of the second domain of complementarity .

[00272] The second complementarity domain can share homology with or be derived from a second naturally occurring complementary domain. In some embodiments, it has at least 50% homology with a second domain of complementarity described in this document, for example, a first domain of complementarity S. pyogenes, S. aureus, N. mening- tidis, or S thermophilus.

[00273] Some or all nucleotides in the second domain of complementarity may have a modification, for example, a modification described in this document. G) Proximal domain

[00274] Examples of proximal domains include those described in WO 2015/161276, for example, in FIGS. 1A-1G on it. In some modalities, the proximal domain is 5 to 20 nucleotides in length. In some embodiments, the proximal domain may share homology with or be derived from a naturally occurring proximal domain. In some embodiments, it has at least 50% homology with a proximal domain in this document, for example, a proximal domain S. pyogenes, S. aureus, N. meningtidis, or S. thermophilus.

[00275] Some or all of the nucleotides in the proximal domain may have a change along the lines described in this document. H) Domain of the tail

[00276] Examples of tail domains include those described in WO 2015/161276, for example, in FIGS. 1A-1G on it. As can be seen from the inspection of the tail domains in WO 2015/161276, for example, in FIG. 1A and FIGS. 1B-1F in it, a wide spectrum of tail domains are suitable for use in gRNA molecules. In several modalities, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In certain embodiments, the tail domain nucleotides are from or share sequence homology of the 5 'end of a naturally occurring tail domain, see, for example, WO 2015/161276, for example, in FIG. 1D or 1E in it. The cause domain also optionally includes sequences that are complementary to each other and that, under at least some physiological conditions, form a duplexed region.

[00277] Tail domains can share homology with or be derived from naturally occurring proximal tail domains. By way of non-limiting example, a given tail domain according to various embodiments of the present invention can share at least 50% homology with a naturally occurring tail domain described in this document, for example, a tail domain S. pyogenes, S. aureus, N. meningtidis, or S. thermophilus.

[00278] In certain cases, the tail domain includes nucleotides at the 3 'end that are related to the in vitro or in vivo transcription method. When a T7 promoter is used for in vitro transcription of the gRNA, these nucleotides can be any nucleotides present before the 3 'end of the DNA standard. When a U6 promoter is used for in vivo transcription, these nucleotides can be the UUUUUU sequence. When alternative pol-III promoters are used, these nucleotides can be various numbers or bases of uracil or they can include alternative bases.

[00279] By means of a non-limiting example, in various modalities of proximal and tail domains, taken together comprise the following sequences:

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCU (SEQ ID NO: 151),

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGGUGC (SEQ ID NO: 152),

AAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGG UGCGGAUC (SEQ ID NO: 153), AAGGCUAGUCCGUUAUCAACUUGAAAAUGUG (SEQ ID NO: 154), AAGGCUAGUCCGUUAUC (SEQ ID: 155)

[00280] In some embodiments, the tail domain comprises the sequence 3 'UUUUUU, for example, if a U6 promoter is used for transcription. In some embodiments, the tail domain comprises the 3 'UUUU sequence, for example, if an H1 promoter is used for transcription. In some embodiments, the tail domain comprises variable numbers of Us 3 ', depending, for example, on the termination signal of the pol-III promoter used. In some modalities, the tail domain comprises the 3 'variable sequence derived from standard DNA if a T7 promoter is used. In some embodiments, the tail domain comprises a 3 'variable sequence derived from standard DNA, for example, whether in vitro transcription is used to generate the RNA molecule. In some embodiments, the tail domain comprises a variable 3 'sequence derived from

Standard DNA, for example, if a pol-II promoter is used to conduct transcription.

[00281] In some modalities, a gRNA has the following structure: 5 '[targeting domain] - [first complementary domain] - [linking domain] - [second complementary domain] - [domain proximal] - [tail domain] -3 'where, the targeting domain comprises a core domain and optionally a secondary domain, and is 10 to 50 nucleotides in length; the first complementarity domain is 5 to 25 nucleotides in length and, in some modalities, it has at least 50, 60, 70, 80, 85, 90, 95, 98 or 99% homology with a first reference complementarity domain in present document described; the binding domain is 1 to 5 nucleotides in length; the proximal domain is 5 to 20 nucleotides in length and, in some modalities, it has at least 50, 60, 70, 80, 85, 90, 95, 98 or 99% homology with a reference domain proximal in the this document described; and the tail domain is absent or a nucleotide sequence is 1 to 50 nucleotides in length and, in some embodiments, has at least 50, 60, 70, 80, 85, 90, 95, 98 or 99% homology with a reference tail domain in this document described. I) exemplary chimeric gRNAs

[00282] In some embodiments, a unimolecular or chemical gRNA comprises, preferably from 5 'to 3': a targeting domain, for example, comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides (which is complementary to a target nucleic acid); a first domain of complementarity; a link domain; a second domain of complementarity (which is complementary to the first domain of complementarity); a proximal domain; and a tail domain, in which (a) the proximal and tail domains, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 3 'nucleotides to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 3 'nucleotides to the last nucleotide of the second complementary domain that is complementary to its nucleotide corresponding to the first complementarity domain.

[00283] In some embodiments, the sequence of (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a gRNA naturally occurring, or with a gRNA described in this document. In some modalities, the proximal and tail domains, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 3 'nucleotides to the last nucleotide of the second complementarity domain. In some modalities, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3 'to the last nucleotide of the second complementary domain that is complementary to its nucleotide corresponding to the first domain of complementarity. In some modalities, the targeting domain comprises, has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (for example, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) that have complementarity with the target domain, for example, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length.

[00284] In some embodiments, the unimolecular or chimeric gRNA molecule (which comprises a targeting domain, a first complementarity domain, a binding domain, a second complementarity domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is described as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us , which serve as a termination signal for the U6 promoter, but which may be absent or in a smaller number:

NNNNNNNNNNNNNNNNNNNNGUUUUAGAGCUAGAAAAUAGCAAG UUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAAGUGGCACC

GAGUCGGUGCUUUUUUAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAAGUGGCACCGAGUCGGUGCUUUUU: (SEQ ID NO 157). In some embodiments, the unimolecular or chimeric gRNA molecule is a S. pyogenes gRNA molecule.

[00285] In some embodiments, the unimolecular or chimeric gRNA molecule (which comprises a targeting domain, a first complementarity domain, a binding domain, a second complementarity domain, a proximal domain and, optionally, a tail domain) comprises the following sequence in which the targeting domain is described as 20 Ns but could be any sequence and range in length from 16 to 26 nucleotides and in which the gRNA sequence is followed by 6 Us , which serve as a termination signal for the U6 promoter, but which may be absent or in smaller numbers:

NNNNNNNNNNNNNNNNNNNNGUUUUAGUACUCUGGAAACAGAAA

UCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUG UUGGCGAGAUUUUUU (SEQ ID NO 158). In some embodiments, the unimolecular or chimeric gRNA molecule is a S. aureus gRNA molecule. The exemplary chimeric gRNA sequences and structures are also shown in WO 2015/161276, for example, in FIGS. 10A-10B on it.

J) exemplary modular gRNAs

[00286] In some embodiments, a modular gRNA comprises the first and second strands. The first tape preferably comprises 5 'to 3'; a targeting domain, for example, comprising 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26 nucleotides; a first domain of complementarity. The second strip comprises, preferably from 5 'to 3': optionally a 5 'extension domain; a second domain of complementarity; a proximal domain; and a tail domain, in which: (a) the proximal and tail domains, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides; (b) there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 3 'nucleotides to the last nucleotide of the second complementarity domain; or (c) there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 3 'nucleotides to the last nucleotide of the second complementary domain that is complementary to its nucleotide corresponding to the first complementarity domain.

[00287] In some embodiments, the sequence of (a), (b), or (c), has at least 60, 75, 80, 85, 90, 95, or 99% homology with the corresponding sequence of a gRNA naturally occurring, or with a gRNA described in this document. In some modalities, the proximal and tail domains, when taken together, comprise at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 nucleotides. In some embodiments, there are at least 15, 18, 20, 25, 30, 31, 35, 40, 45, 49, 50, or 53 3 'nucleotides to the last nucleotide of the second complementarity domain.

[00288] In some modalities, there are at least 16, 19, 21, 26, 31, 32, 36, 41, 46, 50, 51, or 54 nucleotides 3 'to the last nucleotide of the second complementary domain that is complementary to its corresponding nucleotide from the first complement domain

tarity.

[00289] In some modalities, the targeting domain has, or consists of, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides (for example, 16, 17, 18 , 19, 20, 21, 22, 23, 24, 25 or 26 consecutive nucleotides) having complementarity with the target domain, for example, the targeting domain is 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or 26 nucleotides in length. K) Methods for Designating gRNAs

[00290] Methods for designating gRNAs are described in this document, including methods for selecting, designating and validating targeting domains. Exemplary targeting domains are also provided in this document. The targeting domains in this document discussed can be incorporated into the gRNAs described in this document.

[00291] In some embodiments, a guide RNA (gRNA) specific for the target gene (for example, TRAC, TRBC1 and / or TRBC2 in humans) is used for RNA-guided nucleases, for example, Cas, for induce DNA breakdown at a target site or target position. Methods for designating exemplary gRNAs and targeting domains may include those described in, for example, PCT International Publication No. WO 2015/161276. Targeting domains can be incorporated into the gRNA that are used to target Cas9 nuclei to the target site or target position.

[00292] Methods for selecting and validating target sequences, as well as off-target analyzes are described, for example, in Mali et al., 2013 Science 339 (6121): 823-826; Hsu et al. Nat Biotechnol, 31 (9): 827-32; Fu et al., 2014 Nat Biotechnol, doi: 10.1038 / nbt.2808. Pub-Med PMID: 24463574; Heigwer et al., 2014 Nat methods 11 (2): 122-3. doi: 10.1038 / nmeth.2812. PubMed PMID: 24481216; Bae et al., 2014 Bioinformatics PubMed PMID: 24463181; Xiao A et al., 2014 Bioinforma-

matics PubMed PMID: 24389662.

[00293] In some modalities, a software tool may be used to optimize the choice of gRNA within a user's target sequence, for example, to minimize off-target activity across the entire genome. Off-target activity can be different from cleavage. For example, for each possible choice of gRNA using Cas9 from S. pyogenes, software tools can identify all sequences outside the potential target (preceding NAG or NGG PAMs) in the entire genome that contains up to a certain number ( for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) of incompatible base pairs. The cleavage efficiency in each off-target sequence can be predicted, for example, using an experimentally derived weighting scheme. Each possible gRNA can then be classified according to its predicted total off-target cleavage; the best rated gRNAs represent those that are likely to have the highest target and at least off-target cleavage. Other functions, for example, automated reagent design for gRNA vector construction, primer design for the Surfereyor assay on the target, and primer design for high throughput detection and quantification of off-target cleavage through sequencing of next generation, can also be included in the tool. Candidate gRNA molecules can be evaluated by methods known in the art or as described herein.

[00294] In some embodiments, gRNAs for use with Cas9s from S. pyogenes, S. aureus, and N.meningitidis are identified using a DNA sequence search algorithm, for example, using custom gRNA design software based on in the public tool cas-offinder (Bae et al. Bioinformatics. 2014; 30 (10): 1473-1475). Custom gRNA design software scores guides after calculating your projection outside the genome's broad target. Normal-

In particular, matches ranging from perfect matches to 7 incompatibilities are considered for guides ranging in length from 17 to 24. In some respects, since off-target sites are computationally determined, an aggregate score is calculated for each guide and summarized tabular output using a web interface. In addition to identifying potential gRNA sites adjacent to the PAM sequences, the software can also identify all adjacent PAM sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. In some modalities, the genomic DNA sequences for each gene are obtained in the UCSC Genome browser and the sequences can be traced to repeated elements using the publicly available RepeatMasker program. RepeatMasker searches for incoming DNA sequences for repeated elements and regions of low complexity. The output is a detailed note of the repetitions present in a given query sequence.

[00295] After identification, gRNAs can be classified into layers based on one or more of their distance to the target site, their orthogonality and the presence of a 5 'G (based on the identification of close matches in the human genome that contains a Relevant PAM, for example, in the case of S. pyogenes, an NGG PAM, in the case of S. aureus, NNGRR PAM (for example, an NNGRRT or NNGRRV), and in the case of N. meningtidis, an NNNNGATT PAM or NNNNGCTT). Orthogonality refers to the number of sequences in the human genome that contain a minimum number of incompatibilities with the target sequence. A "high level of orthogonality" or "good orthogonality" can, for example, refer to 20-mer targeting domains that do not have identical sequences in the human genome beyond the intended target, nor any sequences that indicate a or two incompatibilities in the target sequence. The targeting domains with good orthogonality are selected to minimize off-target DNA cleavage. It should be understood that this is a non-limiting example and that a variety of strategies can be used to identify gRNAs for use with S. pyo-genes, S. aureus and N. meningitidis or other Cas9 enzymes.

[00296] In some embodiments, gRNAs for use with the S. pyogenes Cas9 can be identified using the publicly available web-based ZiFiT server (Fu et al., Improving CRISPR- Cas nuclease specificity using truncated guide RNAs. Nat Biotechnol 26 January 2014. doi: 10.1038 / nbt.2808. PubMed PMID: 24463574, for original references see Sander et al., 2007, NAR 35: W599-605; Sander et al., 2010, NAR 38: W462 -8). In addition to identifying potential gRNA sites adjacent to the PAM sequences, the software also identifies all adjacent PAM sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. In some respects, the genomic DNA sequences for each gene can be obtained in the UCSC Genome browser and the sequences can be selected for repeated elements using the publicly available Repeat-Masker program. RepeatMasker looks for incoming DNA sequences for repeated elements and regions of low complexity. The output is a detailed note of the repetitions present in a given query sequence.

[00297] After identification, gRNAs for use with a S. pyogenes Cas9 can be classified in layers, for example, in 5 layers. In some modalities, the targeting domains for first layer gRNA molecules are selected based on their distance from the target site, their orthogonality and the presence of a 5 'G (based on the ZiFiT identification of close matches in the human genome that contains an NGG PAM). In some embodiments, both 17-mer and 20-mer gRNAs are designed for targets. In some respects, gRNAs are also selected for both the single gRNA nuclease cut and the double gRNA nickase strategy. Criteria for selecting gRNAs and determining which gRNAs can be used for which strategy can be based on various considerations. In some embodiments, gRNAs for single gRNA nuclease cleavage and for a double gRNA paired "nickase" strategy are identified. In some modalities for selecting gRNAs, including determining which gRNAs can be used for the double gRNA paired nickase strategy, the gRNA pairs must be targeted in the DNA so that the PAMs are facing out and cutting with the Cas9 D10A nickase will result in 5 'overhangs. In some respects, it can be assumed that the cleavage of pairs of dual nickases will result in the deletion of the entire intervening sequence with reasonable frequency. However, cleavage of double nickase pairs can also result in indelible mutations at the site of only one of the gRNAs. The members of the candidate pair can be tested for the efficiency with which they remove the entire sequence compared to just causing indelible mutations at a gRNA site.

[00298] In some embodiments, targeting domains for first layer gRNA molecules can be selected based on (1) a reasonable distance to the target position, for example, within the first 500 bp of downstream coding sequence. start codon, (2) a high level of orthogonality, and (3) the presence of a 5 'G. In some modalities, for the selection of second layer gRNAs, the requirement of 5' G can be removed, but the distance restriction is required and a high level of orthogonality was required. In some embodiments, the third layer selection uses the same distance constraint and the requirement for a 5 'G, but removes the requirement for good orthogonality. In some modalities

the fourth layer selection uses the same distance constraint, but removes the requirement for good orthogonality and starts with a 5 'G. In some embodiments, selecting the fifth layer removes the requirement for good orthogonality and a 5' G, and the longest sequence (for example, the rest of the coding sequence, for example, an additional 500 bp upstream or downstream of the target site of the transcription) is digitized. In certain cases, no gRNA is identified based on the criteria of the particular layer.

[00299] In some embodiments, gRNAs are identified for single gRNA nuclease cleavage, as well as for a double gRNA paired "nickase" strategy.

[00300] In some respects, gRNAs for use with the N. meningitidis and S. aureus Cas9s can be manually identified by genomic DNA sequence scanning for the presence of PAM sequences. These gRNAs can be separated into two layers. In some modalities, for first layer gRNAs, targeting domains are selected within the first 500bp of the coding sequence downstream from the start codon. In some embodiments, for second-layer gRNAs, targeting domains are selected within the preceding coding sequence (downstream of the first 500bp). In certain cases, no gRNA is identified based on the criteria of the particular layer.

[00301] In some embodiments, another strategy to identify guide RNAs (gRNAs) for use with Cas9s of S. pyogenes, S. aureus and N. meningtidis can use a DNA sequence search algorithm. In some respects, the guide RNA design is carried out using custom guide RNA design software based on the public tool cas-offinder (Bae et al. Bioinformatics. 2014; 30 (10): 1473-1475). The aforementioned custom guide RNA design software scores the guides after calculating their broad propensity outside the genome target. Normally, matches ranging from perfect matches to 7 mismatches are considered for guides ranging in length from 17 to 24. Once the sites outside the target are computationally determined, an aggregate score is calculated for each guide and summarized in tabular output using a web interface. In addition to identifying potential gRNA sites adjacent to PAM sequences, the software also identifies all adjacent PAM sequences that differ by 1, 2, 3 or more nucleotides from the selected gRNA sites. In some modalities, the genomic DNA sequence for each gene is obtained from the UCSC genome browser and the sequences are selected for repeating elements using the publicly available RepeatMasker program. RepeatMasker looks for incoming DNA sequences for repeated elements and regions of low complexity. The output is a detailed note of the repetitions present in a given query sequence.

[00302] In some modalities, after identification, gRNAs are classified in layers based on distance to the target site or orthogonality (based on the identification of close matches in the human genome that contains a relevant MAP, for example, in the case of S. pyogenes, an NGG PAM, in the case of S. aureus, NNGRR PAM (for example, an NNGRRT or NNGRRV), and in the case of N. meningtidis, an NNNNGATT or NNNNGCTT PAM. targeting with good orthogonality are selected to minimize cleavage of DNA outside the target.

[00303] As an example, for targets of S. pyogenes and N. miningtidis, 17-mer or 20-mer gRNAs can be designed. As another example, for S. aureus targets, 18-mer, 19-mer gRNAs,

20-mer, 21-mer, 22-mer, 23-mer and 24-mer can be designed.

[00304] In some embodiments, gRNAs for single gRNA nuclease cleavage and for the double gRNA paired "nickase" strategy are identified. In some modalities for selecting gRNAs, including determining which gRNAs can be used for the double gRNA paired nickase strategy, the gRNA pairs must be oriented in the DNA so that the PAMs are facing outward and cutting with the Cas9 D10A nickase will result in 5 'overhangs. In some respects, it can be assumed that the cleavage of pairs of dual niquases will result in the deletion of the entire intervening sequence with reasonable frequency. However, cleavage of double nickase pairs often can also result in indelible mutations at the site of only one of the gRNAs. The members of the candidate pair can be tested for the efficiency with which they remove the entire sequence compared to just causing indelible mutations at the site of a gRNA.

[00305] To design strategies for genetic disruption, in some modalities, the targeting domains for layer 1 gRNA molecules for S. pyogenes are selected based on their distance to the target site and their orthogonality (PAM is NGG). In some cases, targeting domains for layer 1 gRNA molecules are selected based on (1) a reasonable distance from the target position, for example, within the first 500 bp of the downstream coding sequence. start codon and (2) a high level of orthogonality. In some respects, for the selection of layer 2 gRNAs, a high level of orthogonality is not required. In some cases, layer 3 gRNAs remove the requirement for good orthogonality and a longer sequence (for example, the rest of the coding sequence) can be scanned. In certain cases, no gRNA is identified based on the criteria of the particular layer.

[00306] To design strategies for genetic disruption, in some modalities, the targeting domain for layer 1 gRNA molecules for N. meningtidis was selected within the first 500 bp of the coding sequence and had a high level of orthogonality. The targeting domain for N. meningtidis layer 2 gRNA molecules was selected within the first 500 bp of the coding sequence and did not require high orthogonality. The targeting domain for N. meningtidis layer 3 gRNA molecules was selected from the remainder of the 500bp downstream coding sequence. Note that the layers are not inclusive (each gRNA is listed only once). In certain cases, no gRNA was identified based on the criteria of the particular layer.

[00307] To design strategies for genetic disruption, in some modalities, the targeting domain for layer 1 gRNA molecules for S. aureus is selected within the first 500bp of the obtained sequence, has a high level of orthogonality, and contains a NNGRRT PAM. In some embodiments, the targeting domain for layer 2 gRNA molecules for S. au- reus is selected within the first 500bp of the coding sequence, no level of orthogonality is required, and contains an NNGRRT PAM. In some embodiments, the targeting domain for layer 3 gRNA molecules for S. aureus is selected within the remainder of the downstream coding sequence and contains an NNGRRT PAM. In some embodiments, the targeting domain for layer 4 gRNA molecules for S. au- rus is selected within the first 500bp of the coding sequence and contains an NNGRRV PAM. In some embodiments, the targeting domain for layer 5 gRNA molecules for S. aureus is selected from the rest of the coding sequence.

downstream and contains an NNGRRV PAM. In certain cases, no gRNA is identified based on the criteria of the particular layer. 2) Cas9

[00308] Cas9 molecules from a variety of species can be used in the methods and compositions described in this document. Although the Cas9 molecules of S. pyogenes, S. aureus, N. miningitidis, and S. thermophilus are the subject of much of the description in this document, the Cas9 molecules of, derived from, or based on Cas9 proteins from other species listed in this document can be used as well. In other words, although much of the description in this document uses Cas9 molecules from S. pyogenes, S. aureus, N. meningitidis, and S. thermophilus, Cas9 molecules from other species can replace them. Such species include: Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilusdenitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithi, Bacillusporthis, Brillispor, Bacillusporthis, Brillispor. Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidatus puniceispi- rillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matru- chotii, Dinoroseobacter- bacterio- , Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosystusus. eris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp. , Si monsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., Or Vermi- nephrobacter eisenia. Examples of Cas9 molecules can include those described in, for example, WO 2015/161276, WO 2017/193107, WO 2017/093969, US 2016/272999 and US 2015/056705.

[00309] A Cas9 molecule, or Cas9 polypeptide, as the term is used in this document, refers to a molecule or polypeptide that can interact with a gRNA molecule and, together with the gRNA molecule, is located at a site that comprises a target domain and PAM sequence. Cas9 molecule and Cas9 polypeptide, as the terms in this document are used, refer to naturally occurring Cas9 molecules and modified Cas9 molecules (modified), engineered or Cas9 polypeptides that differ, for example, by at least least one amino acid residue, from a reference sequence, for example, the most similar naturally occurring Cas9 molecule.

[00310] Crystal structures were determined for two different naturally occurring bacterial Cas9 molecules (Jinek et al., Science, 343 (6176): 1247997, 2014) and for S. pyogenes Cas9 with a guide RNA (for example, a synthetic fusion of crRNA and TRACrRNA) (Nishimasu et al., Cell, 156: 935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038 / nature13579).

[00311] A naturally occurring Cas9 molecule comprises two lobes: a recognition lobe (REC) and a nuclease lobe (NUC); each of which further comprises domains described in this document. An exemplary scheme for organizing important Cas9 domains in the primary structure is described in WO 2015/161276, for example, in FIGS. 8A-8B on it. The domain nomenclature and numbering of the amino acid residues covered by each domain used throughout this invention is as described in Nishimasu et al. The numbering of the amino acid residues is with reference to Cas9 of S. pyogenes.

[00312] The REC lobe comprises the arginine-rich bridge helix (BH), the REC1 domain and the REC2 domain. The REC lobe does not share structural similarity with other known proteins, indicating that it is a specific functional domain of Cas9. The BH domain is a long α-helix and region rich in arginine and comprises amino acids 60-93 of the Cas9 sequence of S. pyogenes. The domain of REC1 is important for the recognition of repetition: anti-repetition duplex, for example, of a gRNA or a TRACrRNA, and is therefore critical for Cas9's activity, recognizing the target sequence. The REC1 domain comprises two REC1 motifs at amino acids 94 to 179 and 308 to 717 of the S. pyogenes Cas9 sequence. These two REC1 domains, although separated by the REC2 domain in the linear primary structure, come together in the tertiary structure to form the REC1 domain. The REC2 domain, or parts of it, can also play a role in recognizing repetition: anti-repetition duplex. The REC2 domain comprises amino acids 180-307 of the Cas9 sequence of S. pyogenes.

[00313] The NUC lobe comprises the domain of RuvC (also referred to in this document as a domain similar to RuvC), the domain of HNH (also referred to in this document as a domain similar to HH), and the interaction domain of PAM (PI). The RuvC domain shares structural similarity with members of the retroviral integrase superfamily and cleaves a single strand, for example, the non-complementary strand of the target nucleic acid molecule. The domain of

RuvC is assembled from the three divided RuvC motifs (RuvC I, RuvCII, and RuvCIII, which are commonly referred to as the RuvCI domain, or N-terminal RuvC domain, RuvCII domain and RuvCIII domain) at amino acids 1-59 , 718-769, and 909-1098, respectively, of the S. pyogenes Cas9 sequence. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, but in the tertiary structure, the three RuvC motifs group together and form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases, and cleaves a single strand, for example, the complementary strand of the target nucleic acid molecule. The HNH domain is among the motifs of RuvC II-III and comprises amino acids 775-908 of the Cas9 sequence of S. pyogenes. The PI domain interacts with the PAM of the target nucleic acid molecule, and comprises amino acids 1099-1368 of the Cas9 sequence of S. pyogenes. A) A domain similar to RuvC and a domain similar to HNH

[00314] In some embodiments, a Cas9 molecule or Cas9 polypeptide comprises a domain similar to HNH and a domain similar to RuvC. In some modalities, cleavage activity is dependent on the domain similar to RuvC and a domain similar to HNH. A Cas9 molecule or Cas9 polypeptide, for example, an eaCas9 molecule or eaCas9 polypeptide, can comprise one or more of the following domains: a domain similar to RuvC and a domain similar to HNH. In some embodiments, a Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide and the eaCas9 molecule or eaCas9 polypeptide comprises a domain similar to RuvC, for example, a domain similar to RuvC described in this document, and / or a similar domain HNH, for example, a domain similar to HNH described in this document. B) Domains similar to RuvC

[00315] In some embodiments, a domain similar to RuvC cleaves, a single strand, for example, the non-complementary strand of the target nucleic acid molecule. The Cas9 molecule or Cas9 polypeptide can include more than one domain similar to RuvC (for example, one, two, three or more domains similar to RuvC). In some modalities, a domain similar to RuvC is at least 5, 6, 7, 8 amino acids in length, but no more than 20, 19, 18, 17, 16 or 15 amino acids in length. In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises an N-terminal RuvC-like domain of about 10 to 20 amino acids, for example, about 15 amino acids in length. C) Domains similar to RuvC N-Terminals

[00316] Some naturally occurring Cas9 molecules comprise more than one domain similar to RuvC with cleavage being dependent on the domain similar to RuvC N-terminal. Consequently, Cas9 molecules or Cas9 polypeptide may comprise an N-terminal RuvC-like domain.

[00317] In modality, a domain similar to RuvC N-terminal is cleavage-competent.

[00318] In modality, a domain similar to RuvC N-terminal is not competent for cleavage.

[00319] In some embodiments, the domain similar to RuvC N-terminal differs from a sequence of a domain similar to RuvC N-terminal described in this document, for example, in WO 2015/161276, for example, in FIGS. 3A-3B or FIGS. 7A-7B therein, up to 1, but not more than 2, 3, 4, or 5 residues. In some embodiments, 1, 2, or all 3 of the highly conserved residues identified in WO 2015/161276, for example, in FIGS. 3A-3B or FIGS. 7A-7B are present in it.

[00320] In some modalities, the domain similar to RuvC N-

terminal differs from a sequence of a domain similar to RuvC N-terminal in the present document described, for example, in WO 2015/161276, for example, in FIGS. 4A-4B or FIGS. 7A-7B therein, up to 1, but not more than 2, 3, 4, or 5 residues. In some embodiments, 1, 2, 3 or all 4 of the highly conserved residues identified in WO 2015/161276, for example, in FIGS. 4A-4B or FIGS. 7A-7B are present in it. D) Additional RuvC-like domains

[00321] In addition to the RuvC N-terminal domain, the Cas9 molecule or Cas9 polypeptide, for example, an eaCas9 molecule or eaCas9 polypeptide, may comprise one or more additional RuvC-like domains. In some embodiments, the Cas9 molecule or Cas9 polypeptide may comprise two additional domains similar to RuvC. Preferably, the additional domain similar to RuvC is at least 5 amino acids in length and, for example, less than 15 amino acids in length, for example, 5 to 10 amino acids in length, for example, 8 amino acids in length. E) Domains similar to HNH

[00322] In some embodiments, an HNH-like domain cleaves a single-strand complementarity domain, for example, a strand complementary to a double-stranded nucleic acid molecule. In some modalities, a domain similar to HNH is at least 15, 20, 25 amino acids in length, but no more than 40, 35 or 30 amino acids in length, for example, 20 to 35 amino acids in length, for example , 25 to 30 amino acids in length. Exemplary domains similar to HNH are described in this document.

[00323] In some modalities, the domain similar to HNH is competent for cleavage.

[00324] In some modalities, the domain similar to HNH is included

competent cleavage.

[00325] In some embodiments, the domain similar to HNH differs from a sequence of a domain similar to HNH in the present document described, for example, in WO 2015/161276, for example, in FIGS. 5A-5C or FIGS. 7A-7B therein, up to 1, but not more than 2, 3, 4, or 5 residues. In some embodiments, 1 or both of the highly conserved residues identified in WO 2015/161276, for example, in FIGS. 5A-5C or FIGS. 7A-7B are present in it.

[00326] In some embodiments, the domain similar to HNH differs from a sequence of a domain similar to HNH in the present document described, for example, in WO 2015/161276, for example, in FIGS. 6A-6B or FIGS. 7A-7B therein, up to 1, but not more than 2, 3, 4, or 5 residues. In some embodiments, 1, 2, all 3 of the highly conserved residues identified in WO 2015/161276, for example, in FIGS. 6A-6B or FIGS. 7A-7B are present in it. F) Nuclease and Helicase activities

[00327] In some embodiments, the Cas9 molecule or Cas9 polypeptide is able to cleave a target nucleic acid molecule. Normally, wild type Cas9 molecules cleave both strands of a target nucleic acid molecule. Cas9 molecules and Cas9 polypeptides can be modified to alter nuclease cleavage (or other properties), for example, to provide a Cas9 molecule or Cas9 peolypeptide that is a nickase, or that is unable to cleave the target nucleic acid . A Cas9 molecule or Cas9 polypeptide that is able to cleave a target nucleic acid molecule is referred to herein as an eaCas9 molecule or eaCas9 polypeptide.

[00328] In some embodiments, an eaCas9 molecule or an eaCAS9 polypeptide comprises one or more of the following activities

des: a nickase activity, that is, the ability to cleave a single strand, for example, the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double-stranded nuclease activity, that is, the ability to cleave both strands of a double-stranded nucleic acid and create a double-stranded break, which in some embodiments is the presence of two nickase activities; an endonuclease activity; an exonuclease activity; and a helicase activity, that is, the ability to unwind the helical structure of a double-stranded nucleic acid.

[00329] In some embodiments, an enzymatically active molecule or eaCas9 or polypeptide eaCas9 cleaves both strands and results in a double strand break. In some embodiments, an eaCas9 molecule cleaves only one strand, for example, the strand with which the gRNA hybridizes to, or the strand complementary to the strand with which the gRNA hybridizes. In some embodiments, an eaCas9 molecule or polypeptide ea-Cas9 comprises cleavage activity associated with a domain similar to HNH. In some embodiments, an eaCas9 molecule or eaCas9 polypeptide comprises the cleavage activity associated with an N-terminal RuvC-like domain. In some embodiments, an eaCas9 molecule or eaCas9 polypeptide comprises cleavage activity associated with a domain similar to HNH and cleavage activity associated with a domain similar to N-terminal RuvC. In some embodiments, an eaCas9 molecule or an eaCas9 polypeptide comprises a domain similar to active HNH, or competent in cleavage, and a domain similar to RuvC N-terminal inactive or incompetent in cleavage. In some embodiments, an eaCas9 molecule or an eaCas9 polypeptide comprises a domain similar to inactive HNH, or incompetent cleavage, and a domain similar to active or competent N-terminal RuvC cleavage.

[00330] Some Cas9 molecules or Cas9 polypeptides have the

ability to interact with a gRNA molecule, and in conjunction with the gRNA molecule located in a central target domain, but are unable to cleave the target nucleic acid, or are unable to cleave at efficient rates. Cas9 molecules having, or no substantial cleaving activity, are referred to herein as an eiCas9 molecule or eiCas9 polypeptide. For example, an eiCas9 molecule or eiCas9 polypeptide may have no cleavage activity or have substantially less, for example, less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a Cas9 molecule reference or eiCas9 polypeptide, as measured by an assay in this described document. G) Targeting and PAMs

[00331] A Cas9 molecule, or Cas9 polypeptide, is a polypeptide that can interact with a guide RNA (gRNA) molecule and, together with the gRNA molecule, is located at a site that comprises a target domain and a PAM sequence.

[00332] In some embodiments, the ability of an eaCas9 molecule or eaCas9 polypeptide to interact with and cleave a target nucleic acid is dependent on the PAM sequence. The PAM sequence is a sequence in the target nucleic acid. In some embodiments, the cleavage of the target nucleic acid occurs upstream of the PAM sequence. EaCas9 molecules from different bacterial species can recognize different sequence motifs (for example, PAM sequences). In some embodiments, an eaCas9 molecule from S. pyogenes recognizes the sequence motif NGG, NAG, NGA and directs the cleavage of a target nucleic acid sequence 1 to 10, for example 3 to 5, base pairs upstream that sequence. See, for example, Mali et al., Science 2013; 339 (6121): 823-826. In some modalities, an eaCas9 molecule from S. thermophilus recognizes the sequence motif NGGNG and / or NNAGAAW (W = A or T) and directs cleavage.

of a target nucleic acid sequence 1 to 10, for example 3 to 5, base pairs upstream of those sequences.

See, for example, Horvath et al., Science 2010; 327 (5962): 167-170, and Deveau et al., J Bacteriol 2008; 190 (4): 1390-1400. In some embodiments, an eaCas9 molecule from S. mutans recognizes the motif sequence NGG and / or NAAR (R = A or G)) and directs the cleavage of a target nucleic acid sequence from nucleus 1 to 10, for example 3 to 5 pairs of bases, upstream of this sequence.

See, for example, Deveau et al., J Bacteriol 2008; 190 (4): 1390-1400. In some embodiments, a S. aureus eaCas9 molecule recognizes the NNGRR sequence motif (R = A or G) and directs the cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, base pairs a amount of that sequence.

In some embodiments, an eaCas9 molecule from S. aureus recognizes the NNGRRT sequence motif (R = A or G) and directs the cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, base pairs a amount of that sequence.

In some embodiments, a S. au- reus eaCas9 molecule recognizes the NNGRRV sequence motif (R = A or G) and directs the cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, base pairs upstream of that sequence.

In some embodiments, an eaCas9 molecule of N. meningitidis recognizes the sequence motif NNNNGATT or NNNGCTT (R = A or G, V = A, G or C) and directs the cleavage of a target nucleic acid sequence 1 to 10, for example, 3 to 5, base pairs upstream of that sequence.

See, for example, Hou et al., PNAS Early Edition 2013, 1-6. The ability of a Cas9 molecule to recognize a PAM sequence can be determined, for example, using a transformation assay described in Jinek et al., Science 2012 337: 816. In the above mentioned modalities, N can be any nucleotide residue , for example, any of A, G, C or T.

[00333] As discussed in this document, Cas9 molecules can be modified to alter the PAM specificity of the Cas9 molecule.

[00334] Exemplary naturally occurring Cas9 molecules are described in Chylinski et al., RNA Biology 2013 10: 5, 727-737. These Cas9 molecules include Cas9 molecules from a grouped bacterial family 1-78.

[00335] Exemplary naturally occurring Cas9 molecules include a Cas9 molecule from a bacterial family of 1 clustered. Examples include a Cas9 molecule of: S. pyogenes (for example, strain SF370, MGAS10270, MGAS10750, MGAS2096, MGAS315, MGAS5005, MGAS6180, MGAS9429, NZ131 and SSI-1), S. thermophilus (for example, LMD strain -9), S. pseudoporcinus (for example, strain SPIN 20026), S. mutans (for example, strain UA159, NN2025), S. macaacae (for example, strain NCTC11558), S. gallolyticus (for example, strain UCN34, ATCC BAA-2069), S. equines (for example, strain ATCC 9812, MGCS 124), S. dysdalactiae (for example, strain GGS 124), S. bovis (for example, strain ATCC 700338), S. anginosus (for example, strain F0211), S. agalactiae (for example, strain NEM316, A909), Listeria monocytogenes (for example, strain F6854), Listeria innocua (L. innocua, for example, strain Clip11262), Enterococcus italicus ( for example, strain DSM 15952), or Enterococcus faecium (for example, strain 1.231.408). Another exemplary Cas9 molecule is a Cas9 molecule of Neisseria meningitidis (Hou et al., PNAS Early Edition 2013, 1-6).

[00336] In some embodiments, a Cas9 molecule or Cas9 polypeptide, for example, an eaCas9 molecule or ea-Cas9 polypeptide, comprises an amino acid sequence: having 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with; differs by no more than 2, 5, 10, 15, 20, 30, or 40% of the amino acid residues when compared to; differs by at least

in 1, 2, 5, 10 or 20 amino acids, but not more than 100, 80, 70, 60, 50, 40 or 30 amino acids from; or is identical to any Cas9 molecule sequence described herein, or a naturally occurring Cas9 molecule sequence, for example, a Cas9 molecule of a species listed in this document (for example, SEQ ID NOS: 159-162, 227 and 228) or described in Chylinski et al., RNA Biology 2013 10: 5, 727-737; Hou et al., PNAS Early Edition 2013, 1-6. In some embodiments, the Cas9 molecule or Cas9 polypeptide comprises one or more of the following activities: a nickase activity; a double-stranded cleavage activity (for example, an endonuclease and / or exonuclease activity); a helicase activity; or the ability, together with a gRNA molecule, to harbor a target nucleic acid.

[00337] In some embodiments, a Cas9 molecule or Cas9 polypeptide comprises the amino acid sequence of the consensus sequence of WO 2015/161276, for example, in FIGS. 2A-2G in it, where "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, S. thermophilus, S. mutans and L. innocua, and "-" indicates which - or amino acid. In some embodiments, a Cas9 molecule or Cas9 polypeptide differs from the consensus sequence sequence of SEQ ID NOS: 159-162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G in it for at least 1, but no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues. In some embodiments, a Cas9 molecule or Cas9 polypeptide comprises the amino acid sequence of SEQ ID NO: 228 or as described in WO 2015/161276, for example, in FIGS. 7A-7B therein, where "*" indicates any amino acid found in the corresponding position in the amino acid sequence of a Cas9 molecule of S. pyogenes, or N. meningitidis, "-" indicates any amino acid, and "-" indicates any amino acid or absent. In some embodiments, a Cas9 molecule or Cas9 polypeptide differs from the sequence of SEQ ID NO: 227 or 228 or as described in WO 2015/161276, for example, in FIGS. 7A-7B therein at least 1, but not more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues.

[00338] A comparison of the sequence of a number of Cas9 molecules indicates that certain regions are conserved. These are identified in this document as: region 1 (residues 1 to 180, or in the case of region 1 'residues 120 to 180); region 2 (residues 360 to 480); region 3 (residues 660 to 720); region 4 (residues 817 to 900); and region 5 (waste 900 to 960).

[00339] In some embodiments, a Cas9 molecule or Cas9 polypeptide comprises regions 1-5, along with sufficient sequence of additional Cas9 molecule to provide a biologically active molecule, for example, a Cas9 molecule having at least an activity described in this document. In some modalities, each of the regions 1-6, independently, has 50%, 60%, 70%, or 80% homology with the corresponding residues of a Cas9 molecule or Cas9 polypeptide described in this document, for example, established in SEQ ID NOS: 159-162, 227 and 228 or a sequence described in WO 2015/161276, for example, of FIGS. 2A-2G or FIGS. 7A-7B in it. H) Altered or Modified Cas9 Molecules and Cas9 Polypeptides

[00340] Cas9 molecules and Cas9 polypeptides described in this document, for example, naturally occurring Cas9 molecules, may have any of a number of properties, including: nickase activity, nuclease activity (eg endonuclease activity and / or exonuclease); helicase activity; the ability to functionally associate with a gRNA molecule; and the ability to target (or locate to) a site on a nucleic acid (for example, PAM recognition and specificity). In some modalities, a Cas9 molecule or Cas9 polypeptide may include all or a subset of these properties. In typical embodiments, a Cas9 molecule or Cas9 polypeptide has the ability to interact with a gRNA molecule and, in conjunction with the gRNA molecule, locate at a site in a nucleic acid. Other activities, for example, specificity of MAP, cleavage activity, or helicase activity can vary more widely in Cas9 molecules and Cas9 polypeptides.

[00341] Cas9 molecules include modified Cas9 molecules and modified Cas9 polypeptides ("modified", as used in this context, only mean that the Cas9 molecule or Cas9 polypeptide differs from a reference sequence, and does not imply any process or origin limitation). A modified Cas9 molecule or Cas9 polypeptide may comprise altered enzymatic properties, for example, altered nuclease activity (compared to a naturally occurring or reference Cas9 molecule) or altered helicase activity. As discussed in this document, a modified Cas9 molecule or Cas9 polypeptide may have nickase activity (as opposed to double-stranded nuclease activity). In some embodiments, a modified Cas9 molecule or Cas9 polypeptide may have a change that changes its size, for example, an amino acid sequence deletion that reduces its size, for example, with no significant effect on one or more, or any activity from Cas9. In some embodiments, a modified Cas9 molecule or Cas9 polypeptide may comprise an alteration that affects the recognition of MAP. For example, a modified Cas9 molecule can be altered to recognize a PAM sequence other than that recognized by the endogenous wild-type PI domain. In some embodiments, a Cas9 molecule or polypeptide

The Cas9 tide may differ in sequence from a naturally occurring Cas9 molecule, but has no significant change in one or more Cas9 activities.

[00342] Cas9 molecules or Cas9 polypeptides with desired properties can be made in several ways, for example, by changing a Cas9 molecule or parental Cas9 polypeptide, for example, naturally occurring, to provide an altered Cas9 molecule or Cas9 polypeptide having a desired property. For example, one or more mutations or differences from a parent Cas9 molecule, for example, a naturally occurring or modified Cas9 molecule, can be introduced. Such mutations and differences include: substitutions (for example, conservative substitutions or non-essential amino acid substitutions); insertions; or deletions. In some embodiments, a Cas9 molecule or Cas9 polypeptide may comprise one or more mutations or differences, for example, at least 1, 2, 3, 4, 5, 10, 15, 20, 30, 40 or 50 mutations , but less than 200, 100, or 80 mutations relative to a reference, for example, a parental Cas9 molecule.

[00343] In some embodiments, a mutation or mutations does not have a substantial effect on a Cas9 activity, for example, a Cas9 activity described in this document. In some modalities, a mutation or mutations has a substantial effect on a Cas9 activity, for example, a Cas9 activity described in this document. I) Cas9 molecules and modified cleavage and non-cleavage Cas9 polypeptides

[00344] In some embodiments, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, for example, that differs from the naturally occurring Cas9 molecule with the closest homology. For example, a Cas9 molecule or Cas9 polypeptide may differ from naturally occurring Cas9 molecules, for example, a Cas9 molecule of S. pyogenes, as follows: its ability to modulate, for example, decreased or increased, cleavage of a double-stranded nucleic acid (endonuclease and / or exonuclease activity), for example, compared to a naturally occurring Cas9 molecule (for example, a Cas9 molecule from S. pyogenes); its ability to modulate, for example, decreased or increased, cleavage of a single strand of a nucleic acid, for example, a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (activity of nickase), for example, compared to a naturally occurring Cas9 molecule (for example, a Cas9 molecule from S. pyogenes); or the ability to cleave a nucleic acid molecule, for example, a double-stranded or single-stranded nucleic acid molecule, can be eliminated. J) eaCas9 molecules and eaCas9 polypeptides with modified cleavage

[00345] In some embodiments, an eaCas9 molecule or polypeptide eaCas9 comprises one or more of the following activities: cleavage activity associated with a domain similar to N-terminal RuvC; cleavage activity associated with HNH-like domain; cleavage activity associated with HNH-like domain and cleavage activity associated with N-terminal RuvC-like domain.

[00346] In some embodiments, an eaCas9 molecule or an eaCas9 polypeptide comprises a domain similar to active or competent HNH cleavage and a domain similar to inactive or incompetent Nuvial RuvC cleavage. An inactive or incompetent RuvC N-terminal domain similar to exemplary cleavage may have a mutation of an aspartic acid in a domain similar to RuvC N-terminal, for example, an aspartic acid in position 9 of the SEQ ID NOS consensus sequence: 159-162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G or an aspartic acid at position 10 of SEQ ID NO: 228, for example, can be replaced by an alanine. In some modalities, the molecule eaCas9 or polypeptide eaCas9 differs from the wild type in the domain similar to N-terminal RuvC and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, for example, less than 20 , 10, 5, 1 or 0.1% of the cleavage activity of a Cas9 reference molecule, for example, as measured by an assay in this described document. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, for example, a naturally occurring Cas9 molecule, such as a S. pyogenes Cas9 molecule, or S. thermophilus. In some modalities, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.

[00347] In some embodiments, an eaCas9 molecule or an eaCas9 polypeptide comprises an inactive or non-competent HNH domain for cleavage and an active or competent N-terminal RuvC-like domain for cleavage. Exemplary inactive or incompetent HNH-like domains may have a mutation in one or more of: a histidine in a domain similar to HNH, for example, a histidine shown at position 856 of the consensus sequence of SEQ ID NOS: 159- 162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G, for example, can be replaced with an alanine; and one or more asparagines in a HNH-like domain, for example, an asparagine shown at position 870 of the consensus sequence of SEQ ID NOS: 159-162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G and / or at position 879 of the consensus sequence of SEQ ID NOS: 159-162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G, for example, can be replaced with an alanine. In some embodiments, eaCas9 differs from the wild type in the HNH-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, for example, less than 20, 10, 5, 1 or 0.1% the cleavage activity of a reference Cas9 molecule, for example, as measured by an assay in this document. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, for example, a naturally occurring Cas9 molecule, such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology.

[00348] In some embodiments, an eaCas9 molecule or an eaCas9 polypeptide comprises an inactive or unskilled HNH cleavage domain and an active or competent N-terminal RuvC-like cleavage domain. Exemplary inactive or incompetent HNH-like domains may have a mutation in one or more of: a histidine in a domain similar to HNH, for example, a histidine shown at position 856 of the consensus sequence of SEQ ID NOS: 159 -162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G, for example, can be replaced with an alanine; and one or more asparagines in a HNH-like domain, for example, an asparagine shown at position 870 of the consensus sequence of SEQ ID NOS: 159-162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G and / or at position 879 of the consensus sequence of SEQ ID NOS: 159-162, 227 and 228 or the consensus sequence described in WO 2015/161276, for example, in FIGS. 2A-2G, for example, can be replaced with an alanine. In some embodiments, eaCas9 differs from the wild type in the HNH-like domain and does not cleave the target nucleic acid, or cleaves with significantly less efficiency, for example, less than 20, 10, 5, 1 or 0.1% the cleavage activity of a reference Cas9 molecule, for example, as measured by an assay in this document. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, for example, a naturally occurring Cas9 molecule, such as a Cas9 molecule of S. pyogenes, or S. thermophilus. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule having the closest sequence identity or homology. K) Changes in the Ability to Cleave One or Both Tapes of a Target Nucleic Acid

[00349] In some embodiments, exemplary Cas9 activities comprise one or more of MAP specificity, cleavage activity, and helicase activity. A mutation (s) can be present, for example, in: one or more domain similar to RuvC, for example, a domain similar to RuvC N-terminal; a domain similar to HNH; a region outside the RuvC-like domains and the HNH-like domain. In some embodiments, a mutation (s) is present in a domain similar to RuvC, for example, a domain similar to RuvC N-terminal. In some modalities, a mutation (s) is present in a domain similar to HNH. In some embodiments, mutations are present in both a domain similar to RuvC, for example, a domain similar to RuvC N-terminal, and a domain similar to HNH.

[00350] Exemplary mutations that can be made in the RuvC domain or HNH domain with reference to the S. pyogenes sequence include: D10A, E762A, H840A, N854A, N863A and / or D986A.

[00351] In some embodiments, a Cas9 molecule or Cas9 polypeptide is an eiCas9 molecule or eiCas9 polypeptide that comprises one or more differences in a RuvC domain and / or an HNH domain compared to a Cas9 molecule from reference, and the eiCas9 molecule or the eiCas9 polypeptide does not cleave a nucleic acid or cleaves significantly less efficiently than the wild type, for example, when compared to the wild type in a cleavage assay, for example, as described in present document, cuts with less than 50, 25, 10, or 1% of a reference Cas9 molecule, as measured by an assay in this document described.

[00352] Whether or not a particular sequence, for example, a substitution, can affect one or more activity, such as targeting activity, cleavage activity, etc., can be evaluated or predicted, for example, evaluating whether the mutation is conservative. In some embodiments, an "non-essential" amino acid residue, as used in the context of a Cas9 molecule, is a residue that can be changed from a wild type sequence of a Cas9 molecule, for example, a Cas9 molecule naturally occurring, for example, an eaCas9 molecule, without abolishing or more preferably, without substantially altering a Cas9 activity (eg, cleavage activity), while altering an "essential" amino acid residue results in substantial loss of activity (for example, cleavage activity).

[00353] In some embodiments, a Cas9 molecule or Cas9 polypeptide comprises a cleavage property that differs from naturally occurring Cas9 molecules, for example, that differs from the naturally occurring Cas9 molecule with the closest homology. For example, a Cas9 molecule or Cas9 polypeptide may differ from naturally occurring Cas9 molecules, for example, a Cas9 molecule from S aureus, S. pyogenes, or C. jejuni as follows: its ability to modulate, for example example, decreased or increased, cleavage of a double-stranded break (endonuclease and / or exonuclease activity), for example, compared to a naturally occurring Cas9 molecule (for example, a Cas9 molecule from S aureus, S. pyogenes, or C. jejuni); its ability to modulate, for example, decreased or increased, cleavage of a single strand of a nucleic acid, for example, a non-complementary strand of a nucleic acid molecule or a complementary strand of a nucleic acid molecule (activity of nickase), for example, compared to a naturally occurring Cas9 molecule (for example, the Cas9 molecule of S aureus, S. pyogenes, or C. jejuni); or the ability to cleave a nucleic acid molecule, for example, a double-stranded or single-stranded nucleic acid molecule, can be eliminated.

[00354] In some embodiments, the altered Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide comprising one or more of the following activities: cleavage activity associated with a RuvC domain; cleavage activity associated with an HNH domain; cleavage activity associated with an HNH domain and cleavage activity associated with an RuvC domain.

[00355] In some embodiments, the modified Cas9 molecule or Cas9 polypeptide is an eCas9 molecule or an eaCas9 polypeptide that does not cleave a nucleic acid molecule (be it a double or single stranded nucleic acid molecule) or cleaves a molecule of nucleic acid with significantly less efficiency, for example, less than 20, 10, 5, 1 or 0.1% of the cleavage activity of a reference Cas9 molecule, for example, as measured by an assay in the present document described. The reference Cas9 molecule can be a naturally occurring unmodified Cas9 molecule, for example, a naturally occurring Cas9 molecule, such as a Cas9 molecule from S. pyogenes, S. thermophilus, S. aureus, C. jejuni or N. meningitidis. In some embodiments, the reference Cas9 molecule is the naturally occurring Cas9 molecule with the closest sequence identity or homology. In some embodiments, the eiCas9 molecule or eiCas9 polypeptide lacks substantial cleavage activity associated with a RuvC domain and cleavage activity associated with an HNH domain.

[00356] In some embodiments, the modified Cas9 molecule or Cas9 polypeptide is an eaCas9 molecule or eaCas9 polypeptide that comprises the fixed amino acid residues of S. pyogenes shown in the consensus sequence described in WO 2015/161276, for example , in FIGS. 2A-2G, and has one or more amino acids that differ from the S. pyogenes amino acid sequence (for example, has a substitution) in one or more residues (for example, 2, 3, 5, 10, 15, 20 , 30, 50, 70, 80, 90, 100, 200 amino acid residues) in SEQ ID NO: 164 or residue represented by a "-" in the consensus sequence described in WO 2015/161276, for example, in FIGS . 2A-2G.

[00357] In some embodiments, the altered Cas9 molecule or Cas9 polypeptide, for example, an eaCas9 molecule, can be a fusion, for example, of two of the most different Cas9 molecules or Cas9 polypeptides, for example, of two or more naturally occurring Cas9 molecules of different species. For example, a fragment of a naturally occurring Cas9 molecule of a species can be fused to a fragment of a Cas9 molecule of a second species. As an example, a fragment of the Cas9 molecule of S. pyogenes that comprises a domain similar to the N-terminal RuvC can be fused to a fragment of the Cas9 molecule of a species other than S. pyogenes (for example, S. thermophilus) that comprises a domain similar to HNH.

L) Cas9 molecules with altered PAM recognition or no PAM recognition

[00357] Naturally occurring Cas9 molecules can recognize specific PAM sequences, for example, the PAM recognition sequences described in this document for, for example, S. pyogenes, S. thermophilus, S. mutans, S. aureus and N. meningitidis.

[00358] In some embodiments, a Cas9 molecule or Cas9 polypeptide has the same PAM specificities as a naturally occurring Cas9 molecule. In other embodiments, a Cas9 molecule or Cas9 polypeptide has a specificity of PAM not associated with a naturally occurring Cas9 molecule, or a specificity of PAM not associated with a naturally occurring Cas9 molecule to which it has homology nearest sequence. For example, a naturally occurring Cas9 molecule can be altered, for example, to alter PAM recognition, for example, to alter the PAM sequence that the Cas9 molecule or Cas9 polypeptide recognizes to decrease off-target sites and / or improve specificity; or eliminates a PAM recognition requirement. In some embodiments, a Cas9 molecule can be altered, for example, to increase the length of the PAM recognition sequence and / or improve the specificity of Cas9 to a high level of identity, for example, to decrease sites outside target and increase specificity. In some embodiments, the length of the MAP recognition sequence is at least 4, 5, 6, 7, 8, 9, 10, or 15 amino acids in length.

[00359] Cas9 molecules or Cas9 polypeptides that recognize different PAM sequences and / or have reduced off-target activity can be generated using directed evolution. Exemplary methods and systems that can be used for targeted evolution of Cas9 molecules are described, for example, in Esvelt et al. Nature

2011, 472 (7344): 499-503. Cas9 candidate molecules can be evaluated, for example, by methods described in this document.

[00360] Changes in the PI domain, which mediate the recognition of PAM, are discussed in this document. M) Synthetic Cas9 Molecules and Cas9 Polypeptides with Altered PI Domains

[00361] Current genome editing methods are limited in the diversity of target sequences that can be targeted by the PAM sequence that is recognized by the Cas9 molecule used. A synthetic Cas9 molecule (or Syn-Cas9 molecule), or synthetic Cas9 polypeptide (or Syn-Cas9 polypeptide), as the term is used in this document, refers to a Cas9 molecule or Cas9 polypeptide that comprises a domain of Cas9 nucleus of a bacterial species and a functional altered PI domain, ie, a PI domain different from that naturally associated with the Cas9 nucleus domain, for example, of a different bacterial species.

[00362] In some embodiments, the altered PI domain recognizes a PAM sequence that is different from the PAM sequence recognized by the naturally occurring Cas9 from which the Cas9 core domain is derived. In some modalities, the altered PI domain recognizes the same PAM sequence recognized by the naturally occurring Cas9, from which the Cas9 core domain is derived, but with different affinity or specificity. A Syn-Cas9 molecule or Syn-Cas9 polypeptide can be, respectively, a Syn-eaCas9 molecule or Syn-eaCas9 polypeptide or a Syn-eiCas9 molecule or Syn-eiCas9 polypeptide.

An example of a Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises: a) a Cas9 nucleus domain, for example, a Cas9 nucleus domain of S. aureus, S. pyogenes, or C. jejuni; and b)

an altered PI domain of a Cas9 sequence of species X.

[00364] In some embodiments, the RKR motif (the PAM binding motif) of said altered PI domain comprises: differences in 1, 2, or 3 amino acid residues; a difference in the sequence of amino acids in the first, second, or third positions; differences in amino acid sequence in the first and second positions, in the first and third positions, or in the second and third positions; compared to the RKR motif sequence of the native or endogenous PI domain associated with the core Cas9 domain.

[00365] In some embodiments, a Syn-Cas9 molecule or Syn-Cas9 polypeptide can also be optimized in size, for example, the Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises one or more deletions, and optionally one or more ligands arranged between the amino acid residues that flank the deletions. In some embodiments, a Syn-Cas9 molecule or Syn-Cas9 polypeptide comprises a REC deletion. N) Cas9 Molecules and Cas9 Polypeptides Optimized for Size

[00366] Modified Cas9 molecules and modified Cas9 polypeptides described herein include a Cas9 molecule or Cas9 polypeptide that comprises a deletion that reduces the size of the molecule while still retaining the desired Cas9 properties, for example, essentially native conformation , Cas9 nuclease activity, and / or recognition of target nucleic acid molecules. Provided in this document are Cas9 molecules or Cas9 polypeptides that comprise one or more deletions and optionally one or more ligands, in which a ligand is disposed between the amino acid residues that flank the deletion. The methods for identifying suitable deletions in a reference Cas9 molecule, methods for generating Cas9 molecules with a deletion and a linker, and methods for using these Cas9 molecules will be evident in the review of this document.

[00367] A Cas9 molecule, for example, a Cas9 molecule of S. aureus, S. pyogenes, or C. jejuni, having a smaller deletion, for example, has a reduced number of amino acids, than the naturally occurring Cas9 molecule corresponding. The smaller size of the Cas9 molecules allows greater flexibility for release methods and thus increases the utility for editing the genome. A Cas9 molecule or Cas9 polypeptide may comprise one or more deletions that do not substantially affect or decrease the activity of the resulting Cas9 molecules or Cas9 polypeptides described herein. Activities that are retained in Cas9 molecules or Cas9 polypeptides that comprise a deletion as described in this document include one or more of the following: a nickase activity, that is, the ability to cleave a single strand, for example, the non-complementary strand or the complementary strand, of a nucleic acid molecule; a double stranded nuclease activity, that is, the ability to cleave both strands of a double stranded nucleic acid and create a double strand break, which in some embodiments is the presence of two nickase activities; an endonuclease activity; activity of an exonuclease; a helicase activity, that is, the ability to unwind the helical structure of a double-stranded nucleic acid; and recognition activity of a nucleic acid molecule, for example, a target nucleic acid or a gRNA.

[00368] The activity of the Cas9 molecules or Cas9 polypeptides described in this document can be assessed using the activity assays described in this document or are known. O) Identification regions suitable for deletion

[00369] Suitable regions of Cas9 molecules for deletion can be identified by a variety of methods. Naturally occurring orthologous Cas9 molecules of various bacterial species, can be modeled on the crystalline Cas9 structure of S. pyogenes (Nishi- masu et al., Cell, 156: 935-949, 2014) to examine the level of conservation among the Cas9 orthologists selected with respect to the three-dimensional shape of the protein. The less conserved or non-conserved regions that are spatially located distant from the regions involved in Cas9 activity, for example, interface with the target nucleic acid molecule and / or gRNA, represent regions or domains that are candidates for deletion without affecting substance. - increase or decrease the activity of Cas9. P) REC-Optimized Cas9 Molecules or Cas9 Polypeptides

[00370] A REC-optimized Cas9 molecule, or a REC-optimized Cas9 polypeptide, as the term is used in this document, refers to a Cas9 molecule or Cas9 polypeptide that comprises a deletion in one or both of the the REC2 domain and the RE1CT domain (collectively a REC deletion), in which the deletion comprises at least 10% of the amino acid residues in the cognate domain. A REC-optimized Cas9 molecule or Cas9 polypeptide can be an eaCas9 molecule or eaCas9 polypeptide, or an eiCas9 molecule or eiCas9 polypeptide. An example of a REC optimized Cas9 molecule or a REC optimized Cas9 polypeptide comprises: a) a selected deletion from: i) a REC2 deletion; ii) a REC1CT deletion; or iii) a REC1SUB deletion.

[00371] Optionally, a linker is disposed between the amino acid residues that flank the deletion. In some embodiments, a Cas9 molecule or Cas9 polypeptide includes only one deletion or only two deletions. A Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC1CT deletion. A Cas9 molecule or Cas9 polypeptide can comprise a REC2 deletion and a REC1SUB deletion.

[00372] Generally, the deletion will contain at least 10% of the

Naacids in the cognate domain, for example, a REC2 deletion will include at least 10% of the amino acids in the REC2 domain. A deletion can comprise: at least 10, 20, 30, 40, 50, 60, 70, 80, or 90% of the amino acid residues in your cognate domain; all amino acid residues from your cognate domain; an amino acid residue outside its cognate domain; a plurality of amino acid residues outside its cognate domain; the amino acid residue immediately N terminal to its cognate domain; the amino acid residue immediately C terminal to its cognate domain; the amino acid residue immediately N terminal to its cognate and the amino acid residue immediately C terminal to its cognate domain; a plurality of, for example, up to 5, 10, 15, or 20, N-terminal amino acid residues of its cognate domain; a plurality of, for example, up to 5, 10, 15, or 20, amino acid residues C terminating in its cognate domain; a plurality of, for example, up to 5, 10, 15, or 20, amino acid residues N terminal to its cognate domain and a plurality of, for example, up to 5, 10, 15, or 20, amino acid residues C terminals to your cognate domain.

[00373] In some modalities, a deletion does not extend beyond: its cognate domain; the N terminal amino acid residue of its cognate domain; the C terminal amino acid residue of its cognate domain.

[00374] A REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide may include a linker disposed between the amino acid residues that flank the deletion. Binders suitable for use among the amino acid residues that flank a REC deletion in a REC-optimized Cas9 molecule are described in this document.

[00375] In some embodiments, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, except for any REC deletion and associated ligand, has at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100% homology to the naturally occurring Cas9 amino acid sequence, for example, a Cas9 S. aurus molecule, a Cas9 S. pyogenes molecule, or a Cas9 C molecule je- juni.

[00376] In some embodiments, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises a sequence of amino acids that, except for any REC deletion and associated ligand, differs by no more than 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 15, 20, or 25, amino acid residues of the naturally occurring Cas9 amino acid sequence, for example, a Cas9 S. aureus molecule, a Cas9 S. pyogenes molecule , or a Cas9 C. jejuni molecule.

[00377] In some embodiments, a REC-optimized Cas9 molecule or REC-optimized Cas9 polypeptide comprises an amino acid sequence that, except for any REC deletion and associated ligand, differs by no more than 1, 2, 3, 4, 5 , 6, 7, 8, 9, 10, 15, 20, or 25% of the amino acid residues in the amino acid sequence of a naturally occurring Cas9, for example, a Cas9 S. aureus molecule, a Cas9 S. pyogenes molecule, or a Cas9 C. jejuni molecule.

[00378] For sequence comparison, normally a sequence acts as a reference sequence, to which the test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the subsequence coordinates are designated, if necessary, and the program parameters of the sequence algorithm are designated. Standard program parameters can be used, or alternate parameters can be assigned. The sequence comparison algorithm then calculates the percent sequence identities for the test strings against the reference sequence, based on the program parameters. Sequence alignment methods for comparison are well known. The ideal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith and Waterman, (1970) Adv. Appl. Math. 2: 482c, by Needleman and Wunsch's homology alignment algorithm, (1970) J. Mol. Biol. 48: 443, by the search for the similarity method of Pearson and Lipman, (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444, for computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, for example, Brent et al., (2003) Current Protocols in Molecular Biology).

[00379] Two examples of algorithms that are suitable for determining the percentage of sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., (1977) Nuc. Acids Res. 25: 3389-3402; and Altschul et al., (1990) J. Mol. Biol. 215: 403-410, respectively. The software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information.

[00380] The percentage of identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, (1988) Comput. Appl. Biosci. 4: 11-17) that was incorporated into the ALIGN program (version 2.0), using a weight residual table PAM120, a gap length penalty of 12 and a gap penalty of 4. In addition, the identity per - tual between two amino acid sequences can be determined using the algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:

444-453) that was incorporated into the GAP program in the GCG software package (available at www.gcg.com), using a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8 , 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[00381] Sequence information for exemplary REC deletions is provided for 83 naturally occurring Cas9 orthologists described in, for example, International PCT Publication Nos. WO 2015/161276, WO 2017/193107 and WO 2017/093969. Q) Nucleic Acids encoding Cas9 Molecules

[00382] Nucleic acids encoding Cas9 molecules or Cas9 polypeptides, for example, an eaCas9 molecule or eaCas9 polypeptide, can be used in connection with any of the modalities provided in this document.

[00383] Examples of nucleic acids encoding Cas9 molecules or Cas9 polypeptides are described in Cong et al., Science 2013, 399 (6121): 819-823; Wang et al., Cell 2013, 153 (4): 910-918; Mali et al., Science 2013, 399 (6121): 823-826; Jinek et al., Science 2012, 337 (6096): 816-821, and WO 2015/161276, for example, in FIG. 8 on it.

[00384] In some embodiments, a nucleic acid encoding a Cas9 molecule or Cas9 polypeptide may be a synthetic nucleic acid sequence. For example, the synthetic nucleic acid molecule can be modified chemically. In some modalities, the Cas9 mRNA has one or more, for example, all of the following properties: it is buffered, polyadenylated, substituted with 5-methylcytidine and / or pseudouridine.

[00385] In addition, or alternatively, the synthetic nucleic acid sequence can be optimized by codon, for example, at least one non-common codon or less common codon has been replaced by a common codon. For example, synthetic nucleic acid can direct the synthesis of an optimized messenger mRNA, for example,

optimized for expression in a mammalian expression system, for example, described in this document.

[00386] In addition, or alternatively, a nucleic acid that co-encodes a Cas9 molecule or Cas9 polypeptide may comprise a nuclear localization sequence (NLS). Nuclear localization sequences are known.

[00387] If any Cas9 sequences are fused with a C-terminal peptide or polypeptide, it is understood that the stop codon will be removed. R) Other Cas Molecules and Cas Polypeptides

[00388] Various types of Cas molecules or Cas polypeptides can be used to practice the inventions described in this document. In some embodiments, Cas molecules from Type II Cas systems are used. In other embodiments, Cas molecules from other Cas systems are used. For example, Cas Type I or Type III molecules can be used. Exemplary Cas molecules (and Cas systems) are described, for example, in Haft et al., PLoS Computational Biology 2005, 1 (6): e60 and Makarova et al., Nature Review Microbiology 2011, 9: 467-477, the contents both references are incorporated in this document by reference in their entirety. Exemplary Cas molecules (and Cas systems) are also shown in Table 6.

Table 6. Cas Systems Type Name or Subtype Structure Name Families (and Representative- Gene ‡ of the Haft et al.§ coded superfamily protein lia) of pro- (adhesions to the PDB-coded protein) ¶ ficated # * * cas1 • Type I cas1 3GOD, COG1518 SERP2463, • Type II 3LFX and SPy1047 and • Type III 2YZS ygbT cas2 • Type I cas2 2IVY, 2I8E and COG1343 and SERP2462, • Type II 3EXC COG3512 SPy1048, • Type III SPy1723 (Do- terminal domain N) and ygbF cas3 '• Type I ‡‡ cas3 NA COG1203 APE1232 and ygcB cas3' '• Subtype IA NA NA COG2254 APE1231 e • Subtype IB BH0336 cas4 • Subtype IA cas4 and NA COG1468 APE1239 and • Subtype IB csa1 BH0340 Subtype IC • Subtype ID • Subtype II-B cas5 • Subtype IA cas5a, 3KG4 COG1688 APE1234, • Subtype IB cas5d, (RAMP) BH0337, devS • Subtype IC cas5e, and ygcI • Subtype IE cas5h, cas5p, cas5t and cmx5 Subtype IA cas6 and 3I4H COG1583 and PF1131 e • Subtype IB cmx6 COG5551 slr7014 • Subtype ID (RAMP) • Subtype III-A • Subtype III-B cas6e • Subtype IE cse3 1WJ9 (RAMP) ygcH cas6f • Subtype I-F csy4 2XLJ (RAMP) y1727 cas7 • Subtype I-A csa2, csd2, NA COG1857 and devR and ygcJ • Subtype I-B cse4, csh2, COG3649 • Subtype I-C csp1 and cst2 (RAMP)

Type Name or Subtype Structure Name Families (and Representative- Gene ‡ of the Haft et al. System of superfamily encoded protein lia) of pro- (adhesions theine coded for PDB) ¶ stayed # ** cas8a1 • Subtype IA ‡‡ cmx1, cst1, NA Similar to LA3191§§ and csx8, BH0338 PG2018§§ csx13 and CXXC-

CXXC cas8a2 • Subtype IA ‡‡ csa4 e NA PH0918 AF0070, csx9 AF1873, MJ0385, PF0637, PH0918 and SSO1401 cas8b • Subtype IB ‡‡ csh1 and NA Similar to MTH1090 and TM1802 BH0338 TM1802 similar to IC IC1 Subtype IC1 d BH0338 csp2 BH0338 cas9 • Type II ‡‡ csn1 and NA COG3513 FTN_0757 and csx12 SPy1046 cas10 • Type III ‡‡ cmr2, NA COG1353 MTH326, csm1 and Rv2823c§§ and csx11 TM1794§§ cas10d • Subset ID3 G cs3 r3 cs3 r3 cs3 r3 cs3 r3 cs3 r3 cs3 ‡ csy1 • IF subtype csy1 NA Similar to y1724 y1724 csy2 • IF subtype csy2 NA (RAMP) y1725 csy3 • IF subtype csy3 NA (RAMP) y1726 ‡‡ cse1 • Subtype IE cse1 NA YgcL-cse2 Similar ygcL cse2 • a ygcK YgcK csc1 • Subtype ID csc1 NA Similar to alr1563 alr1563 (RAMP) csc2 • Subtype ID csc1 and NA COG1337 slr7012 csc2 (RAMP) csa5 • Subtype IA csa5 NA AF1870 AF1870, MJ0380, PF0643 and SSO1398

Type or subtype name Structure Name Families (and Representative- Gene ‡ of the Haft et al.§ superfamily encoded protein lia) from pro- (adhesions to the PDB-encoded protein) ** stayed # ** csn2 • Subtype II -A csn2 NA Similar to SPy1049 SPy1049 csm2 • Subtype III-A ‡‡ csm2 in the COG1421 MTH1081 and SERP2460 csm3 • Subtype III-A csc2 and NA COG1337 MTH1080 and csm3 (RAMP) SERP2459 csm4 • Subtype III-AG10 NA379 (RAMP) SERP2458 csm5 • Subtype III-A csm5 NA COG1332 MTH1078 e (RAMP) SERP2457 csm6 • Subtype III-A APE2256 and 2WTE COG1517 APE2256 and csm6 SSO1445 cmr1 • Subtype III-B cmr1 NAG3 cmr1 NAG3 -B cmr3 NA COG1769 PF1128 (RAMP) cmr4 • Subtype III-B cmr4 NA COG1336 PF1126 (RAMP) cmr5 • Subtype III-B ‡‡ cmr5 2ZOP and COG3337 MTH324 and 2OEB PF1125 cmr6 • Subtype III-B cmr6 NA1 ) csb1 • IU subtype GSU0053 NA (RAMP) Balac_1306 and GSU0053 csb2 • IU§§ NA subtype (RAMP) Balac_1305 and GSU0054 csb3 • IU NA subtype (RAMP) Balac_1303§§ csx17 • S ubtype IU NA NA NA Btus_2683 csx14 • Subtype IU NA NA NA GSU0052 csx10 • Subtype IU csx10 NA (RAMP) Caur_2274 csx16 • Subtype III-U VVA1548 NA NA VVA1548 csaX • Subtype III-U csaX NA NA SSO1438 cx csx3 NA NA AF1864

Type or subtype name Structure Name Families (and Representative- Gene ‡ of the Haft et al.§ coded superfamily protein lia) from pro- (adhesions to the PDB-coded protein) ¶ stayed # ** csx1 • Subtype III -U csa3, csx1, 1XMX and COG1517 and MJ1666, csx2, 2I71 COG4006 NE0113, DXTHG, PF1127 and NE0113 and TM1812 TIGR0271 0 csx15 • Unknown NA NA TTE2665 TTE2665 csf1 • Type U csf1 • NA type csf1 ) AFE_1039 csf3 • U type csf3 NA (RAMP) AFE_1040 csf4 • U type csf4 NA NA AFE_1037 3) Cpf1

[00389] In some embodiments, the guide RNA or gRNA promotes the specific association targeting of an RNA-guided nuclease, such as a Cas9 or a Cpf1 to a sequence, such as a genomic or episomal sequence in a cell. In general, gRNAs can be unimolecular (which comprises a single RNA molecule, and alternatively referred to as chimeric), or modular (which comprises more than one, and typically two, separate RNA molecules, such as one crRNA and TRACrRNA, which are generally associated with one another, for example, by duplexing). GRNAs and their component parts are described throughout the literature, for example, in Briner et al. (Molecular Cell 56 (2), 333-339, October 23, 2014 (Briner), which is incorporated by reference), and in Cotta-Ramusino.

[00390] Guide RNAs, whether unimolecular or modular, generally include a targeting domain that is totally or partially complementary to a target, and are typically 10-30 nucleotides in length, and in certain embodiments are 16-24 nucleotides in length (for example, 16, 17, 18, 19, 20, 21, 22, 23 or

24 nucleotides in length). In some respects, the targeting domains are at or near the 5 'terminal of the gRNA in the case of a Cas9 gRNA, and at or near the 3' terminal in the case of a Cpf1 gRNA. Although the previous description focused on gRNAs for use with Cas9, it should be appreciated that other RNA-guided nucleases have been (or may in the future be) discovered or invented using gRNAs that differ in some respects from those described to this point. For example, Cpf1 ("CRISPR de Prevotella e Franciscella 1") is a recently discovered RNA-guided nuclease that does not require a TRACrRNA to function. (Zetsche et al., 2015, Cell 163, 759-771, October 22, 2015 (Zetsche I), in the present document incorporated by reference). A gRNA for use in a Cpf1 genome editing system generally includes a targeting domain and a complementarity domain (alternatively referred to as a "handle"). It should also be noted that, in gRNAs for use with Cpf1, the targeting domain is generally present at or near the 3 'end, instead of the 5' end, as shown above in connection with Cas9 gRNAs (the handle is in or near the 5 'end of a Cpf1 gRNA).

[00391] Although there may be structural differences between gRNAs from different prokaryotic species, or between Cpf1 and Cas9 gRNAs, the principles by which gRNAs operate are generally consistent. Due to this consistency of operation, gRNAs can be defined, in general terms, by their targeting domain strings, and skilled technicians will appreciate that a given targeting domain sequence can be incorporated into any suitable gRNA , including a unimolecular or chimeric gRNA, or a gRNA that includes one or more chemical modifications and / or sequential modifications (substitutions, additional nucleotides, truncates, etc.). Thus, in some aspects of this invention, gRNAs can be described only in terms of their targeting domain sequences.

[00392] More generally, some aspects of the present invention relate to systems, methods and compositions that can be implemented using multiple RNA-guided nucleases. Unless otherwise specified, the term gRNA should be understood to encompass any suitable gRNA that can be used with any RNA-guided nuclease, and not just those gRNAs that are compatible with a particular Cas9 or Cpf1 species. By way of illustration, the term gRNA may, in certain embodiments, include a gRNA for use with any RNA-guided nuclease that occurs in a Class 2 CRISPR system, such as a type II or type V CRISPR system, or a nuclease guided by RNA derived or adapted from them.

[00393] Certain exemplary modifications discussed in this section can be included at any position within a gRNA sequence including, without limitation at or near the 5 'end (for example, within 1-10, 1-5, or 1-2 nucleotides of the 5 'end) and / or at or near the 3' end (for example, within 1-10, 1- 5, or 1-2 nucleotides of the 3 'end). In some cases, modifications are positioned within functional motifs, such as, the repetition-anti-repeat duplex of a Cas9 gRNA, a trunk loop structure of a Cas9 or Cpf1 gRNA, and / or a targeting domain of a gRNA.

[00394] RNA-guided nucleases include, but are not limited to, naturally occurring CRISPR Class 2 nucleases, such as Cas9 and Cpf1, as well as other nucleases derived from or obtained from them. In functional terms, RNA-guided nucleases are defined as those nucleases that: (a) interact with (for example, complex with) a gRNA; and (b) together with the gRNA, associated with, and optionally cleaving or modifying, a target region of a DNA that includes (i) a sequence complementary to the gRNA targeting domain and, optionally, (ii) an additional sequence referred to as a "motif adjacent to the protospacer" or "PAM", which is described in more detail below. As the following examples will illustrate, RNA-guided nucleases can be defined, in general terms, by their cleavage activity and PAM specificity, although there may be variations between individual RNA-guided nucleases that share the same specificity. PAM or cleavage activity. Those skilled in the art will appreciate that some aspects of the present invention relate to systems, methods and compositions that can be implemented using any RNA-guided nuclease with a certain specificity of PAM and / or cleavage activity. For this reason, unless otherwise specified, the term RNA-guided nuclease should be understood as a generic term, and not limited to any particular type (eg Cas9 vs. Cpf1), species (eg, S. pyogenes vs. S aureus) or RNA-guided variation (eg, total length vs. truncated or divided; specificity of PAM vs. modified PAM specificity, etc.).

[00395] In addition to recognizing specific sequential orientations of PAMs and protospacers, RNA-guided nucleases in some modalities can also recognize specific PAM sequences. S. aureus cas9, for example, generally recognizes an NNGRRT or NNGRRV PAM sequence, where the N residues are immediately 3 'from the region recognized by the gRNA targeting domain. S. pyogenes cas9 generally recognize NGG PAM sequences. And F. novicida Cpf1 generally recognizes a TTN PAM sequence.

[00396] The Cpf1 crystal structure of Acidaminococcus sp. in complex with crRNA and a double-stranded DNA target (ds) including a TTTN PAM sequence was resolved by Yamano et al. (Cell. May 5, 2016; 165 (4): 949–962 (Yamano), hereby incorporated by reference). Cpf1, like Cas9, has two lobes: a REC (recognition) lobe and a NUC (nuclease) lobe. The REC lobe includes domains of REC1 and REC2, which bear no resemblance to any known protein structures. The NUC lobe, in turn, includes three RuvC domains (RuvC-I, -II and -III) and one BH domain. However, in contrast to Cas9, the REC lobe of Cpf1 lacks an HNH domain, and includes other domains that also bear no resemblance to known protein structures: a structurally unique PI domain, three Wedge (WED) domains (WED- I, -II and - III), and a nuclease domain (Nuc).

[00397] While Cas9 and Cpf1 share similarities in structure and function, it should be appreciated that certain Cpf1 activities are mediated by structural domains that are not analogous to any Cas9 domains. For example, the cleavage of the complementary strand of the target DNA appears to be mediated by the Nuc domain, which differs sequentially and spatially from the Cas9 HNH domain. In addition, the non-targeted portion of Cpf1 gRNA (the handle) adopts a pseudonó structure, instead of a trunk loop structure formed by the repetition duplex: anti-repetition in Cas9 gRNAs.

[00398] Nucleic acids encoding RNA-guided nucleases, for example, Cas9, Cpf1 or functional fragments thereof, are provided herein. Exemplary RNA-guided nucleic acids encoding nuclei have been described previously (see, for example, Cong 2013; Wang 2013; Mali 2013; Jinek 2012).

3. Release of agents for genetic disruption

[00399] In some modalities, the targeted genetic disruption, for example, breaking of DNA, of the endogenous genes that encodes TCR,

such as, TRAC and TRBC1 or TRBC2 in humans, is performed by releasing or introducing one or more agents capable of inducing a genetic disruption, for example, Cas9 and / or gRNA components, to a cell, using any of a series of known delivery methods or vehicle for introduction or transfer to cells, for example using viral delivery vectors, for example, lentiviral, or any of the known methods or vehicles for the release of Cas9 molecules and gRNAs. Exemplary methods are described in, for example, Wang et al. (2012) J. Immunherher. 35 (9): 689-701; Cooper et al. (2003) Blood. 101: 1637–1644; Verhoeyen et al. (2009) Methods Mol Biol. 506: 97-114; and Cavalieri et al. (2003) Blood. 102 (2): 497-505. In some embodiments, nucleic acid sequences that encode one or more components of one or more agents capable of inducing a genetic breakdown, for example, DNA breakdown, are introduced into cells, for example, by any methods to introduce nucleic acids in a cell described in this document or known. In some embodiments, a vector that encodes components of one or more agents capable of inducing a genetic disruption, such as a CRISPR guide RNA and / or a Cas9 enzyme can be released into the cell.

[00400] In some embodiments, one or more agents capable of inducing a genetic disruption, for example, one or more agents that is a Cas9 / gRNA, is introduced into cells as a complex of ri- bonucleoprotein (RNP). RNP complexes include a sequence of ribonucleotides, such as, an RNA or gRNA molecule, and a protein, such as, a Cas9 protein or variant thereof. For example, the Cas9 protein is released as an RNP complex comprising a Cas9 protein and a gRNA molecule targeting the target sequence, for example, using electroporation or another physical release method. In some modalities, the RNP is released in the

squid by electroporation or other physical means, for example, particulate weapon, calcium phosphate transfection, compression or cellular tightening. In some embodiments, RNP can cross a cell's plasma membrane without the need for additional release agents (eg, small molecule agents, lipids, etc.). In some modalities, the release of one or more agents capable of inducing genetic rupture, for example, CRISPR / Cas9, as an RNP offers the advantage that the targeted rupture occurs transiently, for example, in the cells to which an RNP is introduced, without propagation of the agent to cell progenies. For example, RNP release minimizes the agent from being inherited from its progenies, thereby reducing the chance of off-target genetic disruption in the progenies. In such cases, genetic disruption and targeted knockout (discussed later in Section IB) can be inherited by the progeny cells, but without the agent itself, which can still introduce genetic disruption outside the target, being passed over the cells of progeny.

[00401] Agents and components capable of inducing a genetic disruption, for example, a Cas9 molecule and gRNA molecule, can be introduced into target cells in a variety of ways, using a variety of delivery methods and formulations, as established in Tables 7 and 8, or methods described in, for example, WO 2015/161276; US 2015/0056705, US 2016/0272999, US 2017/0211075; or US 2017/0016027. As also described in this document, delivery methods and formulations can be used to deliver standard polynucleotides and / or other agents to the cell in previous or subsequent steps of the methods described in this document.

Table 7. Exemplary Release Methods Elements Molecule (s) Molecule (s) Comments Cas9 gRNA In this modality, a Cas9 molecule and a gRNA are transcribed from DNA. In this

DNA DNA modality, they are encoded in separate molecules. In this modality, a Cas9 molecule and a gRNA are transcribed from DNA, in the

DNA document present a single molecule. In this modality, a Cas9 molecule is transcribed from DNA, and a gRNA is provided

RNA DNA as RNA transcribed or synthesized in vitro In this modality, a Cas9 molecule is translated from mRNA transcribed in vitro, and mRNA RNA a gRNA is supplied as RNA transcribed or synthesized in vitro. In this embodiment, a Cas9 molecule is DNA mRNA translated from mRNA transcribed in vitro, and a gRNA is transcribed from DNA. In this embodiment, a Cas9 molecule is Protein DNA supplied as a protein, and a gRNA is transcribed from DNA. In this embodiment, a Cas9 molecule is provided as a protein, and a Protein RNA gRNA is provided as transcribed or synthesized RNA.

Table 8. Comparison of exemplary release methods Full Length Release- Mode Type / release vector Non-dividing Molecule Exception cells Genome Released physical data (eg electroporation, particle gun) Transit-free acids, Phosphate transfection yes No cell and calcium river, compression c- Lular or squeezing proteins) Retrovirus No Stable yes RNA yes / no Lentivirus yes Stable with Transitó- Adenovirus dif- fications yes No DNA Viral river Viruses Adenoas- yes Stable No associated DNA (AAV) a lot of Varcinia Virus yes Transitó- No DNA rio Herpes Virus yes Stable No DNA Simple Depends on Acids nu- Liposomes Ca- Transitó- yes what is cleic and thionics rio released Proteins Non Viral It depends on Nu- acids Acid- Nanoparticles Transitó- yes on what is kleic and polymer released river Proteins Vehicles Bacteria Attenuate- Transitó- Acids nu- yes No on release of cleic river ration Bacteriophages Transitó- Non-viral modifi cleicos river markets

Release in Integral Duration- Type of Mode / release vector Non-dividing Molecule Exception cells Genome Released biologics Particles- Transitó- Numeric acids to yes virus No mammalian liposomes Bio - logical: fantas- Transitó- Acids nu- yes No but of erythrocyte rio cleicos and Exosomes

[00402] In some embodiments, DNA encoding Cas9 molecules and / or gRNA molecules, or RNP complexes comprising a Cas9 molecule and / or gRNA molecules, can be released into cells by known methods or as described in present document. For example, DNA encoding Cas9 and / or gRNA can be released, for example, by vectors (for example, viral or non-viral vectors), non-vector based methods (for example, using naked DNA or DNA complexes) , or a combination thereof. In some embodiments, the polynucleotide that contains the agents and / or components thereof is released by a vector (for example, viral vector / virus or plasmid). The vector can be any one described in this document.

[00403] In some respects, a CRISPR enzyme (eg, Cas9 nuclease) in combination with (and optionally complexed with) a guide sequence is released into the cell. For example, one or more elements of a CRISPR system are derived from a CRISPR type I, type II, or type III system. For example, one or more elements of a CRISPR system are derived from a specific organism that comprises an endogenous CRISPR system, such as Streptococcus pyogenes, StaphylococcuS aureus or Neisseria menin-

gitides.

[00404] In some embodiments, a Cas9 nuclease (for example, which is encoded by StaphylococcuS aureus or Strepococcus pyogenes mRNA, for example, pCW-Cas9, Addgene # 50661, Wang et al. (2014) Science, 3: 343- 80-4; or lentiviral nuclease or nickase vectors available from Applied Biological Materials (ABM; Canada) such as Cat. No. K002, K003, K005 or K006) and a specific guide RNA for the target gene ( for example, TRAC, TRBC1 and / or TRBC2 in humans) are introduced into cells. In some embodiments, gRNA sequences that are or comprise a targeting domain sequence targeting the target site in a given gene, such as the designated or identified TRAC, TRBC1 and / or TRBC2 genes. A broad genome gRNA database for CRISPR genome editing is publicly available, which contains exemplary sequences of single guide RNA (sgRNA) targeting exons constituting genes in the human or mouse genome (see, for example, genescript. com / gRNA-database.html; see also Sanjana et al. (2014) Nat. methods, 11: 783-4). In some respects, the gRNA sequence is or comprises a sequence with minimal off-target binding to a non-target site or position.

[00405] In some embodiments, the polynucleotide containing the agents and / or components of the same or RNP complex is released by a method based on non-vectors (for example, using naked DNA or DNA complexes). For example, DNA or RNA or proteins or a combination thereof, for example, ribonucleoprotein complexes (RNP), can be released, for example, by organically modified silica or silicate (Ormosil), electroporation, compression or transient cell tightening (for example, as described in Lee, et al. (2012) Nano Lett 12: 6322-27, Kollmannsperger et al (2016) Nat Comm 7, 10372 doi: 10.1038 / ncomms10372), gene weapon, so -

noporation, magnetofection, lipid-mediated transfection, dendrimers, inorganic nanoparticles, calcium phosphates, or a combination thereof.

[00406] In some modalities, the release by means of electroporation comprises the mixture of the cells with the DNA encoding Cas9 and / or gRNA or RNP complex in a cartridge, chamber or cuvette and application of one or more electrical impulses of defined duration and amplitude. In some embodiments, release via electroporation is performed using a system in which cells are mixed with the DNA encoding Cas9 and / or gRNA in a container connected to a device (for example, a pump) that feeds the mixture in a cartridge, chamber or cell in which one or more electrical impulses of defined duration and amplitude are applied, after which the cells are released to a second container.

[00407] In some modalities, the delivery vehicle is a non-viral vector. In some embodiments, the non-viral vector is an inorganic nanoparticle. Exemplary inorganic nanoparticles include, for example, magnetic nanoparticles (for example, Fe3MnO2) and silica. The outer surface of the nanoparticle can be conjugated to a positively charged polymer (for example, polyethyleneimine, polylysine, polyserine) that allows the attachment (for example, conjugation or trapping) of the payload. In some embodiments, the non-viral vector is an organic nanoparticle. Exemplary organic nanoparticles include, for example, SNALP liposomes that contain cationic lipids together with neutral auxiliary lipids that are coated with polyethylene glycol (PEG) and lipid-coated protamine-nucleic acid complexes. Exemplary lipids and / or polymers are known and can be used as modalities provided.

[00408] In some modalities, the vehicle has targeting modifications to increase the target cell update of nano-particles and liposomes, for example, cell specific antigens, monoclonal antibodies, single chain antibodies, aptamers, polymers, sugars and cell-penetrating peptides (for example, described in US 2016/0272999). In some modalities, the vehicle uses fusogenic peptides / polymers and endosome destabilizers. In some modalities, the vehicle undergoes concomitant changes triggered by acid (for example, to accelerate the endosomal space of the load). In some embodiments, a stimulus-cleavable polymer is used, for example, for release into a cell compartment. For example, cationic disulfide polymers that are cleaved in the reducing cell environment can be used.

[00409] In some modalities, the delivery vehicle is a biological non-viral delivery vehicle. In some embodiments, the vehicle is an attenuated bacterium (for example, naturally or artificially modified to be invasive, but attenuated to prevent pathogenesis and express the transgene (for example, Listeria monocytogenes, certain strains of Salmonella, Bifidobacterium longum, and modified Escherichia coli), bacteria with nutritional and tissue-specific tropism for specific target cells, bacteria with surface proteins modified to alter the specificity of the target cell). In some embodiments, the vehicle is a genetically modified bacteriophage (for example, modified phage with high packaging capacity, less immunogenicity, containing mammalian plasmid maintenance sequences and having built-in targeting ligands). In some embodiments, the vehicle is a particle similar to a mammalian virus. For example, modified viral particles can be generated (for example, by purifying the "empty" particles followed by ex vivo assembly of the virus with the desired charge). The vehicle can also be modified to incorporate targeting ligands to alter the specificity of the target tissue. In some embodiments, the vehicle is a biological liposome. For example, the biological liposome is a phospholipid-based particle derived from human cells (eg, erythrocyte ghosts, which are red blood cells broken into spherical structures derived from the individual (eg, tissue targeting can be achieved by binding various tissues or cell-specific ligands), or membrane-bound nanovesicles derived from individual-secretory exosomes (30 -100 nm) of endocytic origin (for example, they can be produced from various types of cells and, therefore, they can be absorbed by cells without the need for targeting ligands).

[00410] In some embodiments, the RNA encoding Cas9 molecules and / or gRNA molecules, can be released into cells, for example, target cells described in this document, by known methods or as described in this document. For example, RNA encoding Cas9 and / or encoding gRNA can be released, for example, by microinjection, electroporation, compression, or transient cell tightening (for example, as described in Lee, et al. (2012) Nano Lett 12 : 6322-27), lipid-mediated transfection, peptide-mediated release, or a combination thereof.

[00411] In some embodiments, the release by means of electroporation comprises the mixture of cells with RNA encoding Cas9 molecules and / or gRNA molecules in a cartridge, chamber or cuvette and applying one or more electrical impulses of duration and amplitude. - all defined. In some embodiments, release via electroporation is performed using a system in which cells are mixed with RNA encoding Cas9 molecules and / or gRNA molecules in a container connected to a device (for example, a pump) which feeds the mixture into a cartridge, chamber or cuvette in which one or more electrical impulses of defined duration and amplitude are applied, after which the cells are released into a second container.

[00412] In some embodiments, Cas9 molecules can be released into cells by known methods or as described in this document. For example, Cas9 protein molecules can be released, for example, by microinjection, electroporation, compression or transient cellular tightening (for example, as a result in Lee, et al. (2012) Nano Lett 12: 6322- 27), lipid-mediated transfection, peptide-mediated release, or a combination thereof. The release can be accompanied by DNA encoding a gRNA or by a gRNA.

[00413] In some modalities, the release by means of electroporation comprises the mixing of cells with Cas9 molecules with or without gRNA molecules in a cartridge, chamber or cuvette and application of one or more electrical impulses of defined duration and amplitude . In some embodiments, release via electroporation is performed using a system in which the cells are mixed with Cas9 molecules with or without gRNA molecules in a container connected to a device (for example, a pump) that feed the mixture into a cartridge, chamber or cuvette in which one or more electrical impulses of defined duration and amplitude are applied, after which the cells are released into a second container.

[00414] In some embodiments, the polynucleotide containing the agents and / or components of the same is released by a combination of a method based on vector and not vector. For example, a virus-sum comprises a liposome combined with an inactivated virus (for example, HIV or influenza virus), which can result in more efficient gene transfer than a viral or liposomal method alone.

[00415] In some embodiments, more than one agent (s) or components of the same are released into the cell. For example, in some embodiments, the agent (s) capable of introducing the genetic disruption of two or more sites in the genome, for example, the TRAC, TRBC1 and / or TRBC2 loci, are released to the cell. In some embodiments, the agent (s) and components of the same (s) are (are) released using a method. For example, in some embodiments, agent (s) to induce genetic disruption of the TRAC, TRBC1 and / or TRBC2 loci is (are) released as polynucleotides encoding the component for genetic disruption. In some embodiments, a polylucleotide can encode agents that target the TRAC, TRBC1 and / or TRBC2 loci. In some embodiments, two or more different polynucleotides can code for agents that target the TRAC, TRBC1 and / or TRBC2 loci. In some modalities, agents capable of introducing genetic disruption can be released as ribonucleoprotein (RNP) complexes, and two or more different RNP complexes can be released together as a mixture, or separately.

[00416] In some embodiments, one or more nucleic acid molecules other than said one or more agents capable of inducing genetic disruption and / or component thereof, for example, the component of the Cas9 molecule and / or the component from the gRNA molecule, such as a standard polynucleotide for HDR-directed integration (such as any standard polynucleotide described in this document, for example, in Section IB), are released. In some modalities, a nucleic acid molecule, for example, a standard polynucleotide, is released at the same time as one or more of the components of the Cas system. In some embodiments, a nucleic acid molecule is released before or after (for example, less than about 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 9 hours, 12 hours, 1 day, 2 days, 3 days, 1 week, 2 weeks, or 4 weeks) that one or more of the components of the Cas system are released. In some embodiments, a nucleic acid molecule, for example, a standard polynucleotide, is released by a medium other than one or more of the components of the Cas system, for example, the component of the Cas9 molecule and / or the component of the molecule of gRNA. The nucleic acid molecule, for example, standard polynucleotide, can be released by any of the release methods described in this document. For example, a nucleic acid molecule, for example, standard polynucleotide, can be released by a viral vector, for example, a retrovirus or a lentivirus, and the component of the Cas9 molecule and / or the component of the gRNA molecule can (m) be released by electroporation. In some embodiments, a nucleic acid molecule, for example, a standard polynucleotide, includes one or more transgenes, for example, transgenes encoding a recombinant TCR, recombinant CAR and / or other gene products. B. Targeted Integration Through Targeted Homology Repair (HDR)

[00417] In some of the modalities provided in this document, homology-directed repair (HDR) can be used for targeted integration of a specific portion of the standard polynucleotide that contains a transgene, for example, nucleic acid sequence encoding a recombinant receptor, at a given location in the genome, for example, the TRAC, TRBC1 and / or TRBC2 locus. In some embodiments, the presence of a genetic disruption (for example, DNA breakdown, as described in Section IA) and a standard polynucleotide that contains one or more homologous arms (for example, that contains sequences of nucleic acid homologous to sequences around a genetic disruption) can induce or direct HDR, with homologous sequences that act as a standard for DNA repair. Based on the homology between the endogenous gene sequence that involves the genetic disruption and the 5 'and / or 3' homology arms included in the standard polynucleotide, cellular DNA repair machinery can use the standard polynucleotide to repair breaking DNA and resynthesizing genetic information at the site of genetic disruption, thereby effectively inserting or integrating transgene sequences into the standard polynucleotide at or near the site of genetic disruption. In some embodiments, genetic disruption, for example, TRAC, TRBC1 and / or TRBC2 locus, can be generated by any of the methods to generate the targeted genetic disruption described in this document.

[00418] Polynucleotides, for example, standard polynucleotides described in this document, are also provided. In some embodiments, the provided polynucleotide can be used in the methods described in this document, for example, involving HDR, to target transgene sequences encoding a portion of a recombinant receptor, for example, recombinant TCR, in endogenous TRAC, TRBC1 and / or TRBC2 locus.

[00419] In some embodiments, the standard polynucleotide is or comprises a polynucleotide that contains a transgene (exogenous or heterologous nucleic acid sequences) encoding a recombinant receptor or a portion of it (for example, one or more strands ( s), recombinant receptor region (s) or domain (s), and homology sequences (for example, homology arms) that are homologous to sequences at or near the endogenous genomic site, for example, at the locus of TRAC, TRBC1 and / or endogenous TRBC2. In some ways, the standard polynucleotide is introduced as a linear DNA fragment or comprised in a vector. In some ways, the step to induce genetic disruption and the step to integrate

Targeted integration (for example, by introducing the standard polynucleotide) are carried out simultaneously or sequentially.

1. Homology-Directed Repair (HDR)

[00420] In some modalities, homologous directed repair (HDR) can be used for targeted integration or insertion of one or more nucleic acid sequences, for example, transgene sequences, in one or more sites (s ) target in the genome, for example, the TRAC, TRBC1 and / or TRBC2 locus. In some embodiments, nuclease-induced HDR can be used to alter a target sequence, integrate a transgene into a particular target site, and / or edit or repair a mutation in a given target gene.

[00421] The change in nucleic acid sequences at the target site can occur by HDR with a standard polynucleotide provided exogenously (also known as donor polynucleotide or standard sequence). For example, the standard polynucleotide provides change in the target sequence, such as insertion of the transgene contained within the standard polynucleotide. In some embodiments, a plasmid or vector can be used as a standard for homologous recombination. In some embodiments, a linear DNA fragment can be used as a standard for homologous recombination. In some embodiments, a single-stranded pattern polynucleotide can be used as a pattern for altering the target sequence by alternative homology-directed repair methods (for example, single-stranded annealing) between the target sequence and the polynucleotide for - drão. The change made by a standard polynucleotide of a target sequence depends on cleavage by a nuclease, for example, a targeted nuclease such as CRISPR / Cas9. Nuclease cleavage may comprise a double-strand break or two single-strand breaks.

[00422] In some embodiments, "recombination" refers to a process of exchanging genetic information between two polynucleotides. In some embodiments, "homologous recombination (HR)" refers to the specialized form of such an exchange that occurs, for example, during the repair of double-strand breaks in cells by means of homology-directed repair mechanisms. This process requires nucleotide sequence homology, uses a standard polynucleotide for the standard repair of a target DNA (that is, the one that experienced double strand rupture, for example, target site in the endogenous gene), and is variously known as "non-crossed gene conversion" or "conversion of short tract gene" as it leads to the transfer of genetic information from the standard polynucleotide to the target. In some embodiments, this transfer may involve the bad correction of the heteroduplex DNA that forms between the broken target and the standard polynucleotide, and / or "synthesis-dependent ribbon ringing", in which the standard polynucleotide is used to resynthesize genetic information. the target, and / or related processes. Such specialized HR often results in a change in the sequence of the target molecule in such a way that part or all of the sequence of the standard polynucleotide is incorporated into the target polynucleotide.

[00423] In some embodiments, a standard polynucleotide, for example, a transgene-containing polynucleotide, is integrated into the genome of a cell through independent homology mechanisms. The methods include creating a double-stranded rupture (DSB) in the genome of a cell and cleaving the standard polynucleotide molecule using a nuclease so that the standard polynucleotide is integrated into the DSB site. In some modalities, the standard polynucleotide is integrated using non-homology-dependent methods (for example, NHEJ). In vivo cleavage, standard polynucleotides can be integrated directly into the genome of a cell at the site of a DSB. The polynucleotide-

The standard video can include one or more of the same target sites for one or more of the nucleases used to create the DSB. In this way, the standard polynucleotide can be cleaved by one or more of the same nucleases used to cleave the endogenous gene into which integration is desired. In some embodiments, the standard polynucleotide includes different target nuclease sites from nucleases used to induce DSB. As described in this document, the genetic disruption of the target site or target position can be created by any mechanisms, such as, ZFNs, TALENs, CRISPR / Cas9 system, or TtAgo nucleases.

[00424] In some embodiments, DNA repair mechanisms can be induced by a nuclease after (1) a single double-strand break, (2) two single-strand breaks, (3) two double-strand breaks with a break occurring on each side of the target site, (4) a double tape break and two single tape breaks with the double tape break and two single tape breaks occurring on each side of the target site (5) four tape breaks single with a pair of single ribbon breaks occurring on each side of the target site, or (6) a single ribbon break. In some embodiments, a standard single-stranded polynucleotide is used and the target site can be altered by alternative HDR.

[00425] The alteration of a target site carried out by a standard polynucleotide depends on cleavage by a nuclease molecule. Nuclease cleavage may comprise a notch, a double strand break, or two single strand breaks, for example, each strand of DNA at the target site. After introducing the ruptures at the target site, resection occurs at the ends of the rupture resulting in regions of DNA protruding from the single strand.

[00426] In canonical HDR, a double-stranded pattern polynucleotide is introduced, comprising the sequence homologous to the target site that will be incorporated directly into the target site or used as a pattern to insert the transgene or correct the sequence of the target site. After resection at rupture, the repair may progress through different pathways, for example, by Holliday's double junction pattern (or double-strand rupture repair, DSBR, pathway) or by strand-dependent ribbon annealing pathway. synthesis (SDSA).

[00427] In the Holliday double junction pattern, the invasion of the ribbon by the two single-ribbon protrusions of the target site occurs to the homologous sequences in the standard polynucleotide, resulting in the formation of an intermediate with two Holliday junctions. The junctions migrate as the new DNA is synthesized from the ends of the invasive strand to fill the gap resulting from resection. The end of the newly synthesized DNA is linked to the dried end, and the junctions are resolved, resulting in insertion at the target site, for example, insertion of the transgene into the standard polynucleotide. The crossing with the standard polynucleotide can occur in the resolution of the junctions.

[00428] In the SDSA pathway, only a single strand protrusion invades the standard polynucleotide and the new DNA is synthesized from the end of the invasive strand to fill the gap resulting from the resection. The newly synthesized DNA then rings with the remaining single strand protrusion, the new DNA is synthesized to fill the gap, and the strands are ligated to produce the modified DNA duplex.

[00429] In alternative HDR, a single-stranded standard polynucleotide, for example, standard polynucleotide, is introduced. A notch, single-strand break, or double-strand break at the target site, to alter a desired target site, is mediated by a nuclease molecule, and resection at the break occurs to reveal protrusions of the single strand. The incorporation of the standard polynucleotide sequence to correct or alter the DNA target site typically occurs via the SDSA pathway, as described in this document.

[00430] "Alternative HDR", or repair directed by alternate homology

ternative, in some embodiments, refers to the process of repairing DNA damage using a homologous nucleic acid (for example, an endogenous homologous sequence, for example, a sister chromatoid, or an exogenous nucleic acid, for example , a standard polynucleotide). Alternative HDR is distinct from canonical HDR, as the process uses different pathways from canonical HDR, and can be inhibited by canonical HDR mediators, RAD51 and BRCA2. In addition, the alternative HDR uses a single-stranded or cut homologous nucleic acid to repair the rupture. "Canonical HDR", or repair directed by canonical homology, in some modalities, refers to the process of repairing DNA damage using a homologous nucleic acid (for example, an endogenous homologous sequence, for example, a sister chromatid, or an exogenous nucleic acid, for example, a standard nucleic acid). Canonical HDR normally acts when there is significant resection in the double strand rupture, forming at least a single strand of DNA. In a normal cell, HDR normally involves a series of steps such as, recognition of a rupture, stabilization of a rupture, resection, single-stranded DNA stabilization, formation of a DNA crossover intermediary, crossing intermediate resolution, and connection. The process requires RAD51 and BRCA2 and the homologous nucleic acid is typically double-stranded. Unless otherwise indicated, the term "HDR" in some embodiments encompasses canonical HDR and alternative HDR.

[00431] In some embodiments, double-stranded cleavage is performed by a nuclease, for example, a Cas9 molecule having cleavage activity associated with a domain similar to HNH and cleavage activity associated with a domain similar to RuvC , for example, a domain similar to RuvC N-terminal, for example, a wild type Cas9. These modalities require only a single gRNA.

[00432] In some modalities, a single, or notched, ribbon break is effected by a nuclease molecule with nickase activity, for example, a Cas9 nickase. DNA cut at the target site can be a substrate for alternative HDR.

[00433] In some embodiments, two single-strand breaks, or notches, are performed by a nuclease, for example, Cas9 molecule, having nickase activity, for example, cleavage activity associated with a domain similar to HNH or cleavage activity associated with a domain similar to RuvC N-terminal. These modalities generally require two gRNAs, one for the placement of each single ribbon break. In some embodiments, the Cas9 molecule having nickase activity cleaves the strand to which the gRNA hybridizes, but not the strand that is complementary to the strand with which the gRNA hybridizes. In some embodiments, the Cas9 molecule having nickase activity does not cleave the strand with which the gRNA hybridizes, but rather cleaves the strand that is complementary to the strand with which the gRNA hybridizes. In some modalities, nickase has HNH activity, for example, a Cas9 molecule having RuvC activity inactivated, for example, a Cas9 molecule having a D10 mutation, for example, the D10A mutation. D10A inactive RuvC; therefore, Cas9 nickase has (only) HNH activity and will cut on the strand to which the gRNA hybridizes (for example, the complementary strand, which does not have the NGG PAM on it). In some modalities, a Cas9 molecule having an H840, for example, an H840A, the mutation can be used as a nickase. H840A inactive HNH; therefore, Cas9 nickase has (only) RuvC activity and cuts on the non-complementary strand (for example, the strand that has the NGG PAM and whose sequence is identical to the gRNA). In some embodiments, the Cas9 molecule is a nickase domain similar to RuvC N-terminal, for example, the Cas9 molecule comprises a mutation in N863, for example, N863A.

[00434] In some modalities, in which a nickase and two gRNAs are used to position two single strand notches, one notch is on the + strand and one notch is on the strand - of the target DNA. PAMs are facing outwards. The gRNAs can be selected so that the gRNAs are separated by, from about 0-50, 0-100, or 0- 200 nucleotides. In some modalities, there is no overlap between the target sequences that are complementary to the targeting domains of the two gRNAs. In some embodiments, gRNAs do not overlap and are separated by up to 50, 100, or 200 nucleotides. In some modalities, the use of two gRNAs can increase specificity, for example, by decreasing the off-target binding (Ran et al., Cell 2013).

[00435] In some modalities, a single notch can be used to induce HDR, for example, alternative HDR. It is contemplated in this document that a single notch can be used to increase the ratio of HR to NHEJ at a given cleavage site, for example, target site. In some embodiments, a single strand break is formed on the DNA strand at the target site to which the targeting domain of the said gRNA is complementary. In another embodiment, a single strand break is formed on the DNA strand at the target site other than the strand which the targeting domain of said gRNA is complementary to.

[00436] In some embodiments, other DNA repair pathways, such as single-stranded (SSA), single-stranded break (SSBR), incompatibility repair (MMR), base excision repair (BER) , nucleotide excision repair (NER), intra-tape cross-linking (ICL), translesion synthesis (TLS), error-free post-replication repair (PRR) can be used by the cell to repair a double-strand or single-strand break created by nucleases.

2. Placement of the Genetic Break (for example, DNA Strand Breaks)

[00437] The targeted integration in the transgene being integrated into a specific gene or locus in the genome. The transgene can be integrated anywhere at or near one of at least one target site or site in the genome. In some embodiments, the transgene is integrated into or near one of at least one target site, for example, within 300, 250, 200, 150, 100, 50, 10, 5, 4, 3, 2, 1 or less base pairs upstream or downstream of the cleavage site, such as within 100, 50, 10, 5, 4, 3, 2, 1 base pairs from each site of the target site, such as, within 50, 10, 5, 4, 3, 2, 1 base pairs on each side of the target site. In some embodiments, the integrated sequence comprising the transgene does not include any vector sequences (for example, viral vector sequences). In some embodiments, the integrated sequence includes a portion of the vector sequences (for example, viral vector sequences).

[00438] The double ribbon break or single ribbon break in one of the tapes must be close enough to the site for targeted integration, so that a change is produced in the desired region, for example, insertion of transgene or correction of a mutation occurs. In some modalities, the distance is not greater than 10, 25, 50, 100, 200, 300, 350, 400 or 500 nucleotides. In some modalities, it is believed that a rupture should be close enough to the site for targeted integration so that the rupture is within the region that is subject to exonuclease-mediated removal during the final resection. In some embodiments, the targeting domain is configured so that a cleavage event, for example, a double-strand or single-strand break, is positioned within 1, 2, 3, 4, 5, 10, 15 , 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400 or 500 nucleotides of the desired region to be changed, for example, site for insertion directed. Rupture,

for example, a double-stranded or single-stranded rupture may be positioned upstream or downstream of the region to be altered, for example, site for targeted insertion. In some modalities, a break is positioned within the desired region to be changed, for example, within a region defined by at least two mutant nucleotides. In some modalities, a rupture is positioned immediately adjacent to the region to be altered, for example, immediately upstream or downstream of the site for targeted integration.

[00439] In some embodiments, a single strand break is accompanied by an additional single strand break, positioned by a second gRNA molecule. For example, targeting domains are configured so that a cleavage event, for example, the two single ribbon breaks, are positioned within 1, 2, 3, 4, 5, 10, 15, 20, 25 , 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 300, 350, 400 or 500 nucleotides from a site for targeted integration. In some embodiments, the first and second gRNA molecules are configured so that, when orienting a Cas9 nickase, a single strand break is accompanied by an additional single strand break, positioned by a second gRNA, sufficiently close to each other to result in a change in the desired region. In some embodiments, the first and second gRNA molecules are configured so that a single strand break positioned by said second gRNA is within 10, 20, 30, 40, or 50 nucleotides from a break positioned by said first gRNA molecule, for example, when Cas9 is a nickase. In some modalities, the two gRNA molecules are configured to position cuts in the same position, or within some nucleotides of each other, on different strips, for example, essentially imitating a double strand break.

[00440] In some modalities, in which a gRNA (unimolecular (or chimeric) or modular gRNA) and Cas9 nuclease induce a double strand rupture with the objective of inducing the insertion of transgene or HDR-mediated correction, the site of cleavage is between 0-200 bp (for example, 0-175, 0 to 150, 0 to 125, 0 to 100, 0 to 75, 0 to 50, 0 to 25, 25 to 200, 25 to 175, 25 to 150, 25 to 125, 25 to 100, 25 to 75, 25 to 50, 50 to 200, 50 to 175, 50 to 150, 50 to 125, 50 to 100, 50 to 75, 75 to 200, 75 to 175, 75 to 150, 75 to 125, 75 to 100 bp) away from the site for targeted integration. In some embodiments, the cleavage site is between 0-100 bp (for example, 0 to 75, 0 to 50, 0 to 25, 25 to 100, 25 to 75, 25 to 50, 50 to 100, 50 to 75 or 75 to 100 bp) away from the site for targeted integration.

[00441] In some modalities, HDR can be promoted using nickases to generate a break with protrusions. In some modes, the single-taped nature of the protrusions may enhance the cell's likelihood of repairing the HDR break as opposed to, for example, NHEJ.

[00442] Specifically, in some modalities, HDR is promoted by selecting a first gRNA that directs a first nickase to a first target site, and a second gRNA that directs a second nickase to a second target site that is on the tape of DNA opposite the first target site and displacement of a first incision. In some embodiments, the target domain of a gRNA molecule is configured to position a cleavage event quite far from a pre-selected nucleotide, for example, the nucleotide of a coding region, so that the nucleotide is not altered. In some embodiments, the target domain of a gRNA molecule is configured to position an intronic cleavage event far enough from an intron / exon edge, or naturally occurring splice signal, to avoid altering the exonic sequence or events unwanted splices. In some modalities, the target domain of a gRNA molecule is configured to position itself in an initial exon, to allow the deletion or knockout of the endogenous gene, and / or to allow integration into the structure of the transgene in or near one of the at least one target site.

[00443] In some embodiments, a double strand break may be accompanied by an additional double strand break, positioned by a second gRNA molecule. In some embodiments, a double strand break may be accompanied by two additional single strand breaks, positioned by a second gRNA molecule and a third gRNA molecule.

[00444] In some modalities, two gRNAs, for example, independently, unimolecular (or chimeric) or modular gRNA, are configured to position a double strand break on both sides of a site for targeted integration.

3. Standard polynucleotides

[00445] A standard polynucleotide having homology with sequences at or near one or more target sites in endogenous DNA can be used to alter the structure of a target DNA, for example, targeted insertion of the transgene. In some embodiments, the standard polynucleotide contains homology sequences (for example, homology arms) flanking the transgene, for example, nucleic acid sequences encoding a recombinant receptor, for targeted insertion. In some embodiments, the homology sequences are directional to the transgene at one or more of the TRAC, TRBC1 and / or TRBC2 loci. In some embodiments, the standard polynucleotide includes additional sequences (coding or non-coding sequences) between the homology arms, such as regulatory sequences, such as promoters and / or enhancers, donor and / or acceptor sites. splice factors, internal ribosome entry site (IRES),

sequences encoding ribosome skip elements (for example, 2A peptides), markers and / or SA sites, and / or one or more additional transgenes.

[00446] The sequence of interest in the standard polynucleotide can comprise one or more sequences that encode a functional polypeptide (e.g., a cDNA), with or without promoter.

[00447] In some embodiments, the transgene contained in the standard polynucleotide comprises a sequence that encodes a cell surface receptor (for example, a recombinant receptor) or a chain thereof, an antibody, an antigen, an enzyme, a factor of growth, the nuclear receptor, a hormone, a lymphokine, a cytokine, a reporter, functional fragments or functional variants and combinations, for example, of any of these descriptions in this document. The transgene can encode one or more proteins useful in cancer therapies, for example, one or more chimeric antigen receptors (CARs) and / or a recombinant T cell receptor (TCR). In some embodiments, the transgene can encode any of the recombinant receptors described in Section IV or any chains, regions and / or domains thereof. In some embodiments, the transgene encodes a recombinant T cell receptor (TCR) or any chains, regions and / or domains therein.

[00448] In certain embodiments, The polynucleotide, for example, the standard polynucleotide contains and / or includes a transgene that encodes a fraction and / or part of a recombinant receptor, for example, a recombinant TCR or a chain of the same . In particular modalities, the transgene is targeted at a target site that is within a gene, locus, or open reading frame that encodes an endogenous receptor, for example, an endogenous TCR gene. In some modalities, the transgene is directed towards integration into the structure within the gene locus, such as resulting in a sequence

coding code encoding a complete, whole, and / or natural-sized receiver.

[00449] In certain embodiments, the standard polynucleotide includes or contains a transgene, a part of a transgene, and / or a nucleic acid encoding the recombinant receptor is a recombinant TCR or strand thereof containing one or more variable domains and one or more constant domains. In certain embodiments, one or more of the recombinant TCR shares complete constant domains, for example, of or about 100% identity, with a constant endogenous TCR domain. In particular modalities, the transgene encodes the part and / or fraction of the recombinant TCR that does not include the constant domain, and the transgene is integrated in structure with the sequence, for example, genomic DNA sequence, which encodes a constant domain of Endogenous TCR. In certain embodiments, the integration results in a coding sequence that encodes the complete, whole, and / or native-sized recombinant TCR or chain. In some embodiments, the coding sequence contains the transgene sequence that encodes a part or fragment of the TCR or strand thereof and an endogenous sequence that encodes an endogenous TCR constant domain.

[00450] In certain embodiments, the recombinant TCR or strand of it contains one or more constant domains that share complete, for example, of or about 100% identity, all or a part and / or fragment of a constant domain of Endogenous TCR. In some embodiments, the transgene encodes a part and / or a fragment of the recombinant receptor that includes a part and / or a fraction of a constant domain, for example, a part or fragment of the constant domain that is completely or partially identical to a constant domain of endogenous TCR. In some modes, the transgene is integrated in structure with the sequence, for example, genomic DNA sequence, which encodes a part and / or fragment of the endogenous TCR constant domain that is not encoded by the transgene. In particular embodiments, the integration results in a coding sequence that encodes the complete, whole and / or natural-sized recombinant TCR or chain and contains the transgene sequence and the endogenous sequence that encodes an endogenous or fragment of the domain contained in the TCR.

[00451] In some embodiments, the transgene encodes a part of a TCR chain, where the part of the TCR chain is smaller than a native, full-size TCR chain. In some modalities, the transgene encodes a part of a TCRα chain that is smaller than a full-length native TCRα chain, for example, a human TCRα chain. In some embodiments, the part of the TCRα chain or includes a TCRα variable domain, for example, a natural-sized TCRα variable domain and a part of a constant TCRα domain. In particular embodiments, the transgene is or contains a sequence of nucleotides that encodes a variable domain of TCR and a part of a sequence of nucleotides that encodes a constant domain of TCRα that is smaller than a natural size of a sequence native nucleotide that encodes a TCRα constant domain. In certain embodiments, the transgene contains a nucleotide sequence that encodes a part of a TCRα chain that is or includes less than 4 exons, 3 complete exons, less than 3 exons, 2 complete exons, less than 2 exons, 1 exon, or less than an exon of a gene, locus, or open reading frame encoding a TCRα domain.

[00452] In certain embodiments, the transgene contains a nucleic acid sequence that encodes a part of a TCR chain, for example, a part of a TCRα chain or a part of a TCRβ chain. In some modalities, the transgene contains a

nucleic acid sequence encoding a part of a TCRα constant domain, for example, a part of a TCRα constant domain or a TCRβ constant domain. In particular embodiments, the nucleotide sequence encoding a TCR constant domain is or is less than 4,600 nucleotides, 4,500 nucleotides,

4,000 nucleotides, 3,500 nucleotides, 3,000 nucleotides, 2,500 nucleotides, 2,000 nucleotides, 1,800 nucleotides, 1,600 nucleotides, 1,500 nucleotides, 1,400 nucleotides, 1,300 nucleotides,

1,200 nucleotides, 1,100 nucleotides, 1,000 nucleotides, 800 nucleotides, 700 nucleotides, 600 nucleotides, 500 nucleotides, 450 nucleotides, 400 nucleotides, 350 nucleotides, 300 nucleotides, 250 nucleotides, 200 nucleotides, 150 nucleotides, 100 nucleotides, or 50 nucleotides in length. In some embodiments, the transgene contains a nucleic acid sequence that encodes the part of the TCR constant domain having, tend to be about, or less than 4,600, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,800, 1,600,

1,500, 1,400, 1,300, 1,200, 1,100, 1,000, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 contiguous nucleotides of a sequence having at least 70%, 75 %, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity with all or part of the nucleic acid sequence set out in SEQ NOS ID: 1, 2, or 3.

[00453] In certain embodiments, the transgene encodes a part of a TCRβ chain that is smaller than a full-length native TCRβ chain, for example, a human TCRβ chain. In some embodiments, the part of the TCRβ chain is or includes a TCRβ variable domain, for example, a natural-sized TCRβ variable domain, and a part of a constant TCRβ domain. In particular modalities, the transgene is or contains a sequence of nucleotides that encodes a variable domain of TCR and a part of a sequence of nucleotides that encodes a constant domain of TCRβ that is smaller than the natural size of a sequence native nucleotide encoding a TCRβ constant domain. In certain embodiments, the transgene contains a sequence of nucleotides that encodes a part of a TCRβ chain that is or includes less than 4 exons, 3 complete exons, less than 3 exons, 2 complete exons, less than 2 exons , 1 exon, or less than an exon of a gene, locus, or open reading frame that encodes a TCRβ domain.

[00454] In some embodiments, the transgene contains a sequence that encodes a part of the TCRα chain and / or a part of a constant domain of TCRα that has been codon optimized. In some embodiments, the transgene contains a sequence that encodes a part of the TCRβ chain and / or a part of a TCRβ constant domain that has been codon-optimized.

[00455] In particular modalities, the transgene does not contain any introns, for example, TRAC, TRBC1, and / or TRBC2 introns, or portions thereof. In some embodiments, the transgene contains a sequence of nucleotides that encodes part of a TCRα chain. In particular embodiments, the nucleotide sequence encoding a part of the TCRα chain does not contain any introns or portions thereof. In certain embodiments, the transgene contains a nucleotide sequence that encodes part of a TCRβ chain. In some embodiments, the sequence of nucleotides that encode a part of the TCRβ chain does not contain any introns.

[00456] In some embodiments, the transgene encodes a part of a TCRα chain with less than 100% amino acid sequence identity to a corresponding part of a native or endogenous TCRα chain. In some embodiments, the part of the TCRα chain contains an amino acid sequence with, with, about, or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or more than 99% identity, but less than 100% identity with a corresponding part of a native or endogenous TCRα chain. In particular embodiments, the transgene encodes a part of a TCRα constant domain with less than 100% amino acid sequence identity with a corresponding part of a native or endogenous TCRα constant domain. In some modalities, the part of the TCRα constant domain contains an amino acid sequence with, with, about, or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% , or greater than 99% identity, but less than 100% identity with a corresponding part of a native or endogenous TCRα chain.

[00457] In particular modalities, the transgene encodes a part of a TCRβ chain with less than 100% amino acid sequence identity with a corresponding part of a native or endogenous TCRβ chain. In certain embodiments, the part of the TCRβ chain contains an amino acid sequence with, with about, or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or greater than 99% identity, but less than 100% identity with a corresponding part of a native or endogenous TCRα chain. In particular embodiments, the transgene encodes a part of a constant domain of TCRβ with less than 100% amino acid sequence identity with a corresponding part of a constant domain of native or endogenous TCRβ. In some modalities, the part of the TCRβ constant domain contains an amino acid sequence with, with, about, or at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% , or greater than 99% identity, but less than 100% identity with a corresponding part of a native or endogenous TCRβ chain.

[00458] In certain embodiments, the transgene contains one or more modifications (s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between a TCRα and TCRβ chain. In some embodiments, the transgene encodes a part of a TCRα that contains a constant domain chain aTCRα that contains a cysteine in a position that corresponds to position 48 with numbering as shown in SEQ ID NO: 24. In some embodiments, the alpha constant domain part contains a part of the TCRα constant domain having an amino acid sequence established in any of SEQ ID NOS: 19 or 24, or an amino acid sequence that has, is about, or has at least 70%, 75%, 80%, 85% 90%, 95%, 97%, 98%, 99% sequence identity thereto, which contains one or more cysteine residues capable of forming a non-native disulfide bond with a TCRβ chain.

[00459] In some embodiments, the transgene encodes a part of a TCRβ chain that contains a part of a constant position TCRβ that contains a cysteine in a position that corresponds to 57 with numbering as mentioned in SEQ ID NO: 20. In some but modalities, the part of the alpha constant domain contains a part of the constant domain TCRα having an amino acid sequence mentioned in any of SEQ ID NOS: 20, 21 or 25, or an amino acid sequence that has, has about, or has at least 70%, 75%, 80%, 85% 90%, 95%, 97%, 98%, 99% sequence identity containing one or more cysteine residues capable of forming a non-native disulfide bond with a chain TCRα.

[00460] In particular modalities, the transgene encodes a part of a TCRα chain and / or a constant domain of TCRα that contains one or more modifications to introduce one or more disulfide bonds. In some embodiments, the transgene encodes a part of a TCRα chain and / or a constant domain of TCRα with one or more modifications to remove or prevent a native disulfide bond, for example, between a TCRα chain and a beta. In some embodiments, one or more native cysteines that form and / or are capable of forming a native interchain disulfide bond are replaced by another residue, for example, serine or alanine. In some embodiments, a portion of the TCRα chain and / or TCRα constant domain is modified to replace one or more non-cysteine residues with a cysteine. In some embodiments, said one or more non-native cysteine residues are capable of forming non-native disulfide bonds, for example, with a TCRβ chain. In some modalities, modalities, cysteine is introduced into one or more of the residue Thr48, Thr45, Tyr10, Thr45, and Ser15 with reference to the numbering of a constant domain TCRα mentioned in SEQ ID NO:

24. In certain modalities, cysteines can be introduced in the residue Ser57, Ser77, Ser17, Asp59, of Glu15 of the TCR β chain with reference to the numbering of the TCRβ chain mentioned in SEQ ID NO: 20. Exemplary non-native disulfide bonds of a TCR are described in published international PCT No. WO 2006/000830, WO 2006/037960, and Kuball et al. (2007) Blood, 109: 2331-2338. In some modalities, the transgene encodes a part of a TCRα chain and / or a constant domain of TCRα that contains one or more modifications to introduce one or more disulfide bonds.

[00461] In some embodiments, the transgene encodes all or part of a TCRα chain and / or a constant domain of TCRα with one or more modifications to remove or prevent a native disulfide bond, for example, between a TCRα chain encoded by the transgene and the endogenous TCRβ chain. In some embodiments, one or more native cysteines that form and / or are capable of forming a native interchain disulfide bond are replaced by another residue, for example, serine or alanine. In some modalities,

a part of the TCRα chain and / or TCRα constant domain is modified to replace one or more non-cysteine residues with a cysteine. In some embodiments, said one or more non-native cysteine residues are capable of forming non-native disulfide bonds, for example, with a TCRβ chain encoded by the transgene. In some embodiments, the transgene encodes all or part of a TCRβ chain and / or a TCRβ constant domain with one or more modifications to remove or prevent a native disulfide bond, for example, between a TCRβ chain encoded by the transgene and the chain Endogenous TCRα. In some embodiments, one or more native cysteines that form and / or are capable of forming a native interchain disulfide bond are replaced by another residue, for example, serine or alanine. In some embodiments, a part of the TCRβ chain and / or TCRβ constant domain is modified to replace one or more non-cysteine residues with a cysteine. In some embodiments, said one or more non-native cysteine residues are capable of forming non-native disulfide bonds, for example, with a TCRα chain encoded by the transgene.

[00462] In some embodiments, one or more different standard polynucleotides are used to direct the integration of the transgene at one or more different target sites. To target integration at different target sites, one or more genetic disruptions (for example, DNA breakdown) are generated at one or more target sites; and one or more different homology sequences are used to direct the integration of the transgene into the respective target site. In some modalities, the transgene inserted at each site is the same or substantially the same. In some modalities, the transgene inserted in each site is different. In some embodiments, two or more different transgenes, which encode two or more different domains or chains of a protein, are inserted into one or more target sites. And bad-

In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or strand thereof encodes a strand of a recombinant TCR and the second transgene encodes a different strand than the recombinant TCR. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or strand thereof encodes the alpha (α) chain of the recombinant TCR and the second transgene encodes the beta (β) chain of the recombinant TCR. In some embodiments, two or more transgenes that encode different domains of the recombinant receptors are targeted for integration into two or more target sites. For example, in some embodiments, the transgene that encodes a recombinant TCRα chain is targeted for integration into the TRAC locus, and the transgene that encodes a recombinant TCRβ chain is targeted for integration into the TRBC1 and / or TRBC2 loci.

[00463] In some modalities, the one or more target sites are at or close to one or more of the TRAC, TRBC1 and / or TRBC2 loci. In some embodiments, the first target site is close to the coding sequence of the locus of the TRAC gene, and the second target site is close to the coding sequence of the locus of the TRBC1 gene. In some embodiments, the first target site is close to the coding sequence of the locus of the TRAC gene, and the second target site is close to the coding sequence of the locus of the TRBC2 gene. In some embodiments, the first target site is close to the coding sequence for the TRAC gene locus, and the second target site is both the TRBC1 and TRBC2 loci, for example, in a sequence that is conserved between TRBC1 and TRBC2.

[00464] In some modalities, one or more different DNA sites, for example, the TRAC, TRBC1 and / or TRBC2 loci, are targeted, and one or more transgenes are inserted at each site. In some modalities, the transgene inserted at each site is the same or substantially the same. In some modalities, the transgene inserted in each site is different. In some embodiments, a transgene is inserted into only one of the target sites (for example, the TRAC locus), and another target site is targeted for editing the gene (for example, knockout).

[00465] In some embodiments, any of the lengths and positions of the homology arms and position relative to the target sites, as in the present document described, can also be applied to one or more second standard polynucleotides.

[00466] In some embodiments, nuclease-induced HDR results in the insertion of a transgene (also called "exogenous sequence" or "transgene sequence") for the expression of a transgene for targeted insertion. The sequence of the standard polynucleotide is typically not identical to the genomic sequence where it is placed. A standard sequence polynucleotide can contain a non-homologous sequence flanked by two regions of homology to allow efficient HDR at the location of interest. In addition, the standard polynucleotide sequence may comprise a vector molecule that contains sequences that are not homologous to the region of interest in the cell chromatin. A standard polynucleotide sequence may contain several discontinuous regions of homology to cell chromatin. For example, for the targeted insertion of sequences not normally present in a region of interest, said sequences can be present in a transgene and flanked by regions of homology with the sequence in the region of interest.

[00467] Polynucleotides for insertion can also be referred to as polynucleotides or "transgene" molecules or "exogenous sequences" or "donors. The standard polynucleotide can be DNA, single-stranded and / or double-stranded and can be introduced into a cell in a linear or circular shape. See also North American Patent Publication

americana nº 20100047805 and 20110207221. The standard polynucleotide can also be introduced in the form of DNA, which can be introduced into the cell in a circular or linear fashion. If introduced linearly, the ends of the standard polynucleotide can be protected (for example, from exonucleolytic degradation) by any known methods. For example, one or more dideoxynucleotide residues are added to the 3 'terminus of a linear molecule and / or self-complementing oligonucleotides are attached to one or both ends. See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA 84: 4959-4963; Nehls et al. (1996) Science 272: 886-889. Additional methods to protect exogenous polynucleotides from degradation include, but are not limited to, addition of terminal amino group (s) and the use of modified internucleotide bonds, such as phosphorothioates, phosphoramidates, and O-methyl residues rhinosis or deoxyribose. If introduced as a double strand, the standard polynucleotide can include one or more nuclease target sites, for example, nuclease target sites flanking the transgene to be integrated into the cell genome. See, for example, North American Patent Publication No. 20130326645.

[00468] In some embodiments, the standard double-stranded polynucleotide includes sequences (for example, coding sequences, also called transgene) with a length greater than 1 kb, for example between 2 and 200 kb, between 2 and 10 kb (or any value between them). The standard double-stranded polynucleotide also includes at least one nuclease target site, for example. In some embodiments, the standard polynucleotide includes at least 2 target sites, for example, for a pair of ZFNs or TALENs. Typically, the nuclease target sites are outside the sequence of transgenes, for example, 5 'and / or 3' to the transgene sequences, for cleavage of the transgene. The nuclease cleavage sites can be for any nucleases. In some embodiments, the nuclease target sites contained in the double stranded standard polynucleotide are for the same nucleases used to cleave the endogenous target into which the cleaved standard polynucleotide is integrated by independent homology methods.

[00469] In some embodiments, the standard nucleic acid system is double-stranded. In some embodiments, the standard nucleic acid system is single-stranded. In some embodiments, the standard nucleic acid system comprises a single strand part and a double strand part.

[00470] In some embodiments, the standard polynucleotide contains the transgene, for example, nucleic acid sequences encoding the recombinant receptor, flanked by homology sequences (also called "homology arms") at the 5 'and 3' ends, to allow the DNA repair mechanism, for example, homologous recombination mechanisms, to use the standard polynucleotide as a repair pattern, effectively inserting the transgene into the target site of integration in the genome. The homology arm should extend at least as far as the region where the final resection can take place, for example, in order to allow the resected single strip protrusion to find a complementary region within the standard polynucleotide. The total length can be limited by parameters such as the size of the plasmid or viral packaging limits. In some modalities, a homology arm does not extend into repeated elements, for example, ALU repetitions or LINE repetitions.

[00471] Exemplary homology arm lengths include at least or at least about 50, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900, 1000, 2000, 3000, 4000 , or 5000 nucleotides. In some embodiments, the length of the homology arm is 50-100, 100-250, 250-500, 500-750, 750-1000, 1000-2000, 2000-3000, 3000-4000, or 4000-5000 nucleotides.

[00472] In some embodiments, one or more of the homology arms refuse a sequence of nucleotides that encode a part of a TCR, for example, a constant domain of TCR such as an alpha or beta TCR. In some modalities, one or more arms of homology are connected or linked in structure with the transgene. Thus, in some embodiments, one or more of the homology arms and the transgene encodes a part of a TCR chain that is larger than the part of the TCR chain encoded only by the transgene. In some embodiments, the combination of one or more of the homology arms and the transgene encodes a complete exon of a gene, locus, or open reading frame that encodes a TCR constant domain, for example, a TCRα or TCRβ constant domain. In some embodiments, one or more homology arms contain a nucleotide sequence that encodes all or part of an intron, for example, a TRAC, TRBC1, or TRBC2 intron.

[00473] The target site (also known as "target position", "target DNA sequence" or "target location"), in some modalities, refers to a site in a target DNA (for example, chromosome) which is modified by said one or more agents capable of inducing a genetic disruption, for example, a Cas9 molecule. For example, the target site may be a cleavage of the modified Cas9 molecule of DNA at the target site and targeted modification of the standard polynucleotide, for example, targeted insertion of the transgene, at the target site. In some embodiments, a target site can be a site between two nucleotides, for example, adjacent nucleotides, in the DNA where one or more nucleotides are added. The target site can comprise one or more nucleotides that are altered by a standard polynucleotide. In some embodiments, the target site is within the target sequence (for example, a sequence to which the gRNA binds).

In some embodiments, a target site is upstream or downstream of a target sequence (for example, a sequence to which the gRNA binds). In some respects, a pair of single tape breaks (eg, incisions) on each side of the target site can be generated.

[00474] In some embodiments, the standard polynucleotide comprises about 500 to 1000, for example, 600 to 900 or 700 to 800, base pairs of homology on either side in the endogenous gene. In some embodiments, the standard polynucleotide comprises about 500, 600, 700, 800, 900 or 1000 base pairs of homology 5 'from the target site, 3' from the target site, or both 5 'and 3' from the target site. In certain embodiments, the target site is within the TRAC, TRBC1, and / or TRBC2 gene, locus or open reading frame.

[00475] In some embodiments, the standard polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000 , 4000, or 5000 base pairs of 3 'homology of the target site. In some embodiments, the standard polynucleotide comprises about 100 to 500, 200 to 400 or 250 to 350, 3 'base pairs of the transgene and / or target site. In some embodiments, the standard polynucleotide comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs of 5 'homology to the target site. In some modalities, the target site is within the TRAC, TRBC1, and / or TRBC2 gene, locus or open reading structure.

[00476] In some embodiments, a standard polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 base pairs of 5 'homology to the target site. In some embodiments, the standard polynucleotide comprises about 100 to 500, 200 to 400 or 250 to 350, 5 'base pairs of the transgene and / or target site. In some embodiments, the standard polynucleotide comprises less than about 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, or 10 base pairs of 3 'homology to the target site. In particular embodiments, the target site is within the TRAC, TRBC1, and / or TRBC2 gene, locus or open reading frame.

[00477] In some embodiments, a standard polynucleotide is a nucleic acid sequence that can be used in conjunction with one or more agents capable of introducing a genetic disruption to alter the structure of a target site. In some embodiments, the target site is modified to have part or all of the standard polynucleotide sequence, typically at or near the cleavage sites. In some embodiments, the standard polynucleotide is single-stranded. In some embodiments, the standard polynucleotide is double-stranded. In some embodiments, the standard polynucleotide is DNA, for example, double-stranded DNA. In some embodiments, the standard polynucleotide is single-stranded DNA. In some modalities, the standard polynucleotide is encoded in the same main chain as the vector, for example, AAV genome, plasmid DNA, such as Cas9 and gRNA. In some embodiments, the standard polynucleotide is excised from a vector's main chain in vivo, for example, flanked by gRNA recognition sequences. In some embodiments, the standard polynucleotide is on a separate polynucleotide molecule such as Cas9 and gRNA. In some embodiments, Cas9 and gRNA are introduced as a ribonucleoprotein complex (RNP), and the standard polynucleotide is introduced as a polynucleotide molecule, for example, in a vector.

[00478] In some modalities, the standard polynucleotide alters the structure of the target site, for example, the insertion of the transgene, participating in a repair event directed to homology. In some embodiments, the standard polynucleotide changes the sequence of the target site.

[00479] In some embodiments, the standard polynucleotide includes a sequence that corresponds to a site in the target sequence that is cleaved by one or more agents capable of introducing a genetic disruption. In some embodiments, the standard polynucleotide includes a sequence that corresponds to both, a first site in the target sequence that is cleaved into a first agent capable of introducing a genetic disruption, and a second site in the target sequence that is cleaved in a second of the agent capable of introducing a genetic disruption.

[00480] A standard polynucleotide comprises the following components: [5 'homology arm] - [transgene] - [3' homology arm]. The homology arms provide recombination on the chromosome, thereby inserting the transgene into the DNA at or near the cleavage site, for example, target sites. In some modalities, the homology arms flank the most distal cleavage sites.

[00481] In some embodiments, the 3 'end of the homology arm 5' is the position close to the 5 'end of the transgene. In some forms, the 5 'homology arm may extend by, at, about, or at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides 5 'from the 5' end of the transgene.

[00482] In some embodiments, the 5 'end of the homology arm 3' is the position close to the 3 'end of the transgene. In some embodiments, the 3 'homology arm can extend at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000 , 4000, or 5000 nucleotides 3 'from the 3' end of the transgene.

[00483] In some embodiments, for targeted insertion, the homology arms, for example, the 5 'and 3' homology arms, can each comprise about 1000 base pairs (bp) of sequence flanking the most distal gRNAs ( for example, 1000 bp of sequence on each side of the mutation).

[00484] In some embodiments, one or more second standard polynucleotides comprising one or more second transgenes can be introduced. In some modalities, the referred one or more second transgenes are directed to integration in or close to one of at least one target site by means of repair directed to homology (HDR).

[00485] In some embodiments, said one or more second standard polynucleotides comprise the structure [second 5 'homology arm] - [one or more second transgenes] - [second 3' homology arm]. The homology arms provide recombination on the chromosome, thereby inserting the transgene into the DNA at or next cleavage site, for example, target sites. In some modalities, the homology arms flank the most distal cleavage sites. In some embodiments, the second 5 'homology arm and the second 3' homology arm comprise nucleic acid sequences homologous to the nucleic acid sequences around the at least one target site. In some embodiments, the second 5 'homology arm comprises nucleic acid sequences that are homologous to the second 5' nucleic acid sequences of the target site. In some embodiments, the second 3 'homology arm comprises nucleic acid sequences that are homologous to the second 3' nucleic acid sequences at the target site. In some modalities, the second 5 'homology arm and the second 3' homology arm independently are at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs, or less than or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs. In some embodiments, the second 5 'homology arm and the second 3' homology arm independently are between about 50 and 100, 100 and 250, 250 and 500,

500 and 750, 750 and 1000, 1000 and 2000 base pairs. In some modes, the second 5 'homology arm and the second 3' homology arm independently are about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800 , 900, 1000, 1500, or 2000 base pairs.

[00486] In some modalities, said one or more second transgenes are targeted for integration at or near the target site in the TRAC gene. In some embodiments, said one or more second transgenes are targeted for integration at or near the target site in the TRBC1 or TRBC2 gene.

[00487] It is contemplated that one or both homology arms can be shortened to avoid the inclusion of certain elements of sequence repetition, for example, Alu repetitions or LINE elements. For example, a 5 'homology arm can be shortened to avoid a sequence repetition element. In some instances, a 3 'homology arm may be shortened to avoid a sequence repetition element. In some embodiments, both the 5 'and 3' homology arms may be shortened to avoid including certain elements of sequence repetition. It is contemplated in the present document that standard polynucleotides for targeted insertion can be designed for use as a single-stranded oligo-nucleotide, for example, a single-stranded oligodeoxynucleotide (ssODN). When using an ssODN, the 5 'and 3' homology arms can be up to about 200 base pairs (bp) in length, for example, at least 25, 50, 75, 100, 125, 150, 175, or 200 bp in length. Longer homology arms are also contemplated for ssODNs as improvements in oligonucleotide synthesis continue to be made. In some embodiments, a longer homology arm is made by a method other than chemical synthesis, for example, by denaturing a long double-stranded nucleic acid and purifying one of the strands, for example, by affinity for a specific strand sequence anchored to a solid substrate.

[00488] In some embodiments, the alternative HDR proceeds more efficiently when the standard polynucleotide extended the 5 'homology to the target site (ie, in the 5' direction of the target site ribbon). As a result, in some embodiments, the standard polynucleotide has a longer homology arm and a shorter homology arm, in which the longest homology arm can ring 5 'from the target site. In some ways, the arm that can ring 5 'to the target site is at least 25, 50, 75, 100, 125, 150, 175, or 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 3000, 4000, or 5000 nucleotides from the target site or the 5 'or 3' end of the transgene. In some modalities, the arm that can ring 5 'at the target site is at least 10%, 20%, 30%, 40% or 50% longer than the arm that can ring 3' at the site target target. In some embodiments, the arm that can ring 5 'to the target site is at least 2x, 3x, 4x, or 5x longer than the arm that can ring 3' to the target site. Depending on whether an ssDNA pattern can ring with the intact ribbon or the target site ribbon, the homology arm that rings 5 'to the target site may be at the 5' end of the ssDNA pattern or the 3 'end of the ssDNA, respectively.

Similarly, in some embodiments, the standard polynucleotide has a 5 'homology arm, a transgene, and a 3' homology arm, so that the standard polynucleotide contains homology extended to 5 'of the target site. For example, the 5 'homology arm and the 3' homology arm may be of substantially the same length, however, the transgene may extend 5 'further from the target site than 3' from the target site. In some embodiments, the homology arm extends at least 10%, 20%, 30%, 40%, 50%, 2x, 3x, 4x, or 5x beyond the 5 'end of the target site than the 3 end '

of the target site.

[00490] In some embodiments, the alternative HDR proceeds more efficiently when the standard polynucleotide is centered on the target site. As a result, in some embodiments, the standard polynucleotide has two homology arms that are essentially the same size.

[00491] For example, the first homology arm of a standard polynucleotide can have a length within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the second homology arm of the standard polynucleotide.

[00492] Similarly, in some embodiments, the standard polynucleotide has a 5 'homology arm, a transgene, and a 3' homology arm, so that the standard polynucleotide extends substantially the same distance on both sides of the target site. For example, the homology arms can be of different lengths, however, the transgene can be selected to compensate for this. For example, the transgene may extend more 5 'from the target site than 3' from the target site, however, the 5 'homology arm of the target site is shorter than the 3' homology arm of the target site, to compensate . The reverse is also possible, for example, that the transgene may extend more 3 'from the target site than 5' from the target site, however, the homology arm 3 'of the target site is shorter than the 5 'homology arm of the target site, to compensate.

[00493] In some embodiments, the standard polynucleotide is a single-stranded nucleic acid. In another embodiment, the standard polynucleotide is a double-stranded nucleic acid. In some embodiments, the standard polynucleotide comprises a sequence of nucleotides, for example, from one or more nucleotides, which will be added to or pattern a change in the target DNA. In some embodiments, the standard polynucleotide comprises a sequence of nucleotides that can be used to modify the target site. In some modalities, the standard polynucleotide comprises a nucleotide sequence, for example, of one or more nucleotides, which corresponds to the wild type sequence of the target DNA, for example, of the target site.

[00494] The standard polynucleotide can comprise a transgene. In some embodiments, the standard polynucleotide comprises a 5 'homology arm. In some embodiments, the standard nucleic acid comprises a 3 'homology arm.

[00495] In some embodiments, the standard polynucleotide is linear double-stranded DNA. The length can be, for example, about 200-5000 base pairs, for example, about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 base pairs. The length can be, for example, at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 base pairs. In some ways, the length is not greater than 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 base pairs . In some embodiments, a standard double-stranded polynucleotide has a length of about 160 base pairs, for example, about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800- 1800, 900-1700, 1000-1600, 1100-1500 or 1200-1400 base pairs.

[00496] The standard polynucleotide can be single-stranded linear DNA In some embodiments, the standard polynucleotide is (i) single-stranded linear DNA that can ring the strand of target DNA, (ii) single-stranded DNA that can ring the intact strand of the target DNA, (iii) single-stranded linear DNA that can ring the transcribed strand of the target DNA, (iv) single-stranded linear DNA that can ring the non-transcribed DNA strand target, or more than one of the previous ones.

[00497] The length can be, for example, about 200-5000 base pairs, for example, about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800 , 2000, 2500, 3000, 4000 or 5000 nucleotides. The length can be, for example, at least 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some modalities, the length does not exceed 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000 or 5000 nucleotides. In some embodiments, a single-stranded standard polynucleotide has a length of about 160 nucleotides, for example, about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800-1800, 900-1700, 1000-1600, 1100-1500 or 1200-1400 nucleotides.

[00498] In some embodiments, the standard polynucleotide is circular double-stranded DNA, for example, a plasmid.

[00499] In some embodiments, the standard polynucleotide comprises about 500 to 1000 base pairs of homology on each side of the transgene and / or the target site. In some forms, the standard polynucleotide comprises about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs. 5 'homology of the target site or transgene, 3' of the target site or transgene, or both 5 'and 3' of the target site or transgene. In some modalities, the standard polynucleotide comprises at least 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs. 5 'homology of the target or transgene site, 3' of the target or transgene site, or both 5 'and 3' of the target or transgene site. In some embodiments, the standard polynucleotide comprises no more than 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of homology 5 'from the target or transgene site, 3' from the target or transgene site, or both 5 'and 3' from the target or transgene site.

[00500] In some embodiments, the length of any of the polynucleotides, for example, standard polynucleotides, can be, for example, or about 200-10000 nucleotides, for example, or about 200, 300, 400, 500 , 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 nucleotides, or a value between any of the above. In some embodiments, the length may be, for example, at least about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000, 6000, 7000, 8000, 9000 or 10,000 nucleotides, or a value between any of the above. In some embodiments, the length is no more than or about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1200, 1400, 1600, 1800, 2000, 2500, 3000, 4000, 5000 , 6000, 7000, 8000, 9000 or 10,000 nucleotides. In some embodiments, the length is about 200-4000, 300-3500, 400-3000, 500-2500, 600-2000, 700-1900, 800-1800, 900-1700, 1000-1600, 1100 -1500 or 1200-1400 nucleotides. In some embodiments, the polynucleotide is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4,250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10,000 nucleotides in length, or any value between any of the above. In some embodiments, the polynucleotide is between or about 2,500 and about or about 5,000 nucleotides, about or about 3,500 and about or 4,500 nucleotides, or about or about 3,750 nucleotides and about or 4,250 nucleotides in length. In some embodiments, the polynucleotide is about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4,250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10000 nucleotides in length.

[00501] In some embodiments, the standard polynucleotide contains homology arms to target the endogenous TRAC locus (se-

exemplary nucleotide sequence of the human TRAC gene locus shown in SEQ ID NO: 1; NCBI Reference Sequence: NG_001332.3, TRAC). In some embodiments, a genetic rupture of the locus of TRAC is introduced in the initial coding region of the gene, including sequence immediately after a transcription start site, within a first exon of the coding sequence, or within 500 bp of the starting site of the transcript (for example, less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp), or within 500 bp of the starting codon (for example, less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp). In some embodiments, a genetic disruption is introduced using any of the nucleases and / or gRNAs targeted in this document described, for example, in Section I.A. In some embodiments, the standard polynucleotide comprises about 500 to 1000, for example, 600 to 900 or 700 to 800, base pairs of homology on both sides of a genetic disruption introduced by targeted nucleases and / or gRNAs. In some embodiments, the standard polynucleotide comprises about 500, 600, 700, 800, 900 or 1000 base pairs of sequences from the 5 'homology arm, which is homologous to 500, 600, 700, 800, 900 or 1000 base pairs of 5 'sequences of the genetic disruption (for example, at the TRAC locus), the transgene, and about 500, 600, 700, 800, 900 or 1000 base pairs of 3' homology arm sequences , which is homologous to 500, 600, 700, 800, 900 or 1000 base pairs of the 3 'sequences of the genetic disruption (for example, at the TRAC locus).

[00502] In some embodiments, the standard polynucleotide contains homology arms to target the intravenous TRBC1 or TRBC2 locus (exemplary nucleotide sequence of the human TRBC1 gene locus shown in SEQ ID NO: 2; Reference Sequence NCBI: NG_001333.2, TRBC1; exemplary nucleotide sequence of the TRBC2 gene locus shown in SEQ ID NO: 3; NCBI Reference Sequence: NG_001333.2, TRBC2). In some embodiments, a genetic disruption of the TRBC1 or TRBC2 locus is introduced into the initial coding region of the gene, including the sequence immediately after a transcription start site, within a first exon of the coding sequence, or within 500 bp from the transcription start site (e.g. less than 500, 450, 400, 350, 300, 250, 200, 150, 100, or 50 bp), or within 500 bp of the start codon (e.g., less than 500, 450, 400, 350, 300, 250, 200, 150, 100 or 50 bp). In some embodiments, genetic disruption is introduced using any of the nucleases and / or gRNAs targeted in this document described, for example, in Section I.A. In some embodiments, the standard polynucleotide comprises about 500 to 1000, for example, 600 to 900 or 700 to 800, base pairs of homology on both sides of the genetic disruption introduced by targeted nucleases and / or gRNAs. In some embodiments, the standard polynucleotide comprises about 500, 600, 700, 800, 900 or 1000 base pairs of sequences from the 5 'homology arm, which is homologous to 500, 600, 700, 800, 900 or 1000 base pairs of 5 'sequences of the genetic disruption (for example, at the TRBC1 or TRBC2 locus), the transgene, and about 500, 600, 700, 800, 900 or 1000 base pairs of homology arm sequences 3 ', which is homologous to 500, 600, 700, 800, 900 or 1000 base pairs of the 3' sequences of the genetic disruption (for example, at the TRBC1 or TRBC2 locus).

[00503] In some embodiments, any of the lengths and positions of the homology arms and position relative to the target site (s), such as any described in this document, may also apply to one or more second standard polynucleotide (s).

[00504] In some cases, the standard polynucleotide comprises a promoter, for example, a promoter that is exogenous and / or is not present at or near the target locus. In some embodiments, the promoter directs expression only on a specific cell type (for example, a T cell or B cell or a specific NK cell promoter). In some modalities in which the coding sequences of the functional polypeptide have no promoter, the expression of the integrated transgene is then ensured by transcription conducted by an endogenous promoter or other control element in the region of interest.

[00505] The transgene, including the transgene encoding the recombinant receptor or antigen-binding portion of the same or a chain of the same and / or the one or more second transgene, can be inserted so that its expression is conducted by the endogenous promoter at the integration site, namely the promoter that drives the expression of the endogenous gene into which the transgene is inserted (for example, TRAC, TRBC1 and / or TRBC2). For example, coding sequences in the transgene can be inserted without a promoter, however, in the structure with the coding sequence of the endogenous target gene, so that the expression of the integrated transgene is controlled by the transcription of the promoter. endogenous at the integration site. In some embodiments, the transgene encoding the recombinant TCR or antigen-binding fragment or strand thereof and / or the one or more second transgene is independently operably linked to the endogenous promoter of the gene at the target site. In some embodiments, a ribosome skipping element / autoclipping element, such as a 2A element, is placed in the amount of the transgene coding sequence, so that the ribosome skipping element / autocleaving element is placed. placed in the structure with the endogenous gene.

[00506] In some embodiments, the transgene encodes a portion of a TCR having a chain of TCRα or portion thereof, and a chain of TCRβ or part thereof. In some embodiments, the encoded TCRα chain and the TCRβ chain are separated by a linker or a spacer region. In some embodiments, a linker sequence is included that links the TCRα and TCRβ chains to form the single polypeptide strand. In some embodiments, the ligand is long enough to cover the distance between the C-terminal of the α chain and the N-terminal of the β chain, or vice versa, while ensuring that the length of the ligand is not as long to the point of blocking or reducing binding to a target peptide-MHC complex. In some embodiments, the linker can be any linker capable of forming a single polypeptide strand, while maintaining the specificity of TCR binding. In some modalities, the linker may contain from or about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acid residues, for example, 29, 30, 31 or 32 amino acids. In some modalities, the linker has the formula -PGGG- (SGGGG) n-P-, where n is 5 or 6 and P is proline, G is glycine and S is serine (SEQ ID NO: 22). In some embodiments, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 23). In some embodiments, the linker or spacer between the TCRα chain or portion thereof and a TCRβ chain or portion thereof that is recognized by and / or is capable of being cleaved by a protease. In certain embodiments, the linker or spacer between the TCRα chain or portion thereof and a TCRβ chain or portion thereof contains a ribosome skip element or a self-cleaving element.

[00507] In some embodiments, the transgene is or includes a nucleotide sequence that is or includes the structure [TCRβ chain] - [Linker] - [TCRα chain portion]. In particular embodiments, the transgene is or includes a sequence of nucleotides that is or includes the structure [TCRβ chain] - [autocleaving element] - [portion of the TCRα chain]. In certain embodiments, the transgene is or includes a sequence of nucleotides that is or includes the structure [TCRβ chain] - [ribosome bounce sequence] - [TCRα chain portion]. In some embodiments, the transgene is or includes a nucleotide sequence that is or includes the structure [TCRα chain] - [linker] - [portion of the TCRβ chain]. In particular embodiments, the transgene is or includes a sequence of nucleotides that is or includes the structure [TCRα chain] - [autocleaving element] - [TCRβ chain portion]. In certain embodiments, the transgene is or includes a nucleotide sequence that is or includes the structure [TCRα chain] - [ribosome bounce sequence] - [TCRβ chain portion]. In some embodiments, the structures are encoded by a single or double-stranded polynucleotide polynucleotide strip, in a 5 'to 3' orientation.

[00508] In some cases, the ribosome jump element / autocleaving element, such as a T2A, can cause the ribosome to jump (ribosome jump) the synthesis of a peptide bond at the C-terminus of an element 2A, leading to the separation between the end of the 2A sequence and the next downstream peptide (see, for example, Felipe, Genetic Vaccines and Ther. 2:13 (2004) and Felipe et al. Traffic 5: 616- 626 (2004)). This allows the inserted transgene to be controlled by the transcription of the endogenous promoter at the integration site, for example, TRAC, TRBC1 and / or TRBC2 promoter. Exemplary ribosome skipping elements / autocleaving elements include foot and mouth disease virus 2A sequences (F2A, for example, SEQ ID NO: 11), equine rhinitis A virus (E2A, for example, SEQ ID NO : 10), Thosea asigna virus (T2A, for example, SEQ ID NO: 6 or 7), and porcine teschovirus-1 (P2A, for example, SEQ ID NO: 8 or 9), as described in the Northern Patent Publication -American

No. 20070116690. In some embodiments, the standard polynucleotide includes a P2A ribosome skip element (sequence shown in SEQ ID NO: 8 or 9) upstream of the transgene, for example, recombinant receptor encoding nucleic acids.

[00509] In some embodiments, the transgene may comprise a promoter and / or enhancer, for example, a constitutive promoter or an inducible or tissue-specific promoter. In some modalities, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, the early promoter of simian virus 40 (SV40), the immediate cytomegalovirus (CMV) early promoter, the human Ubiquitin C (UBC) promoter, the promoter of human elongation factor 1α (EF1α), the mouse phosphoglycerate kinase 1 (PGK) promoter, and chicken β-actin promoter coupled to the early CMV enhancer (CAGG). In some embodiments, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter, a synthetic promoter that contains the U3 region of a MoMuLV LTR modified with myoproliferative sarcoma virus enhancer (sequence shown in SEQ ID NO: 18 or 126; see, Challita et al. (1995) J. Virol. 69 (2): 748-755). In some modes, the promoter is a tissue-specific promoter. In another mode, the promoter is a viral promoter. In another embodiment, the promoter is a non-viral promoter. In some cases, the promoter is selected from among the promoter of human elongation factor 1 alpha (EF1α) (sequence shown in SEQ ID NO: 4 or 5) or a modified form of it (EF1α promoter with HTLV1 enhancer; if - sequence shown in SEQ ID NO: 127) or the MND promoter (sequence shown in SEQ ID NO: 18 or 126). In some ways, the transgene does not include a regulatory element, for example, a promoter.

[00510] In some modalities, a "tandem" cassette is integrated into the selected site.

In some embodiments, one or more of the "tandem" cassettes encode one or more polypeptides or factors, each of which is independently controlled by a regulatory element or all controlled as a multicistronic expression system.

In some embodiments, such as those where the polynucleotide contains a first and a second nucleic acid sequence, the coding sequences that encode each of the different polypeptide chains can be operably linked to a promoter, which can be the same or different.

In some embodiments, a nucleic acid molecule may contain a promoter that drives the expression of two or more different polypeptide chains.

In some embodiments, such nucleic acid molecules can be multicistronic (bicistronic or tristristronic, see, for example, North American Patent No. 6,060,273). In some modalities, the transcription units can be modified as a bicistronic unit that contains an IRES (internal ribosome entry site), which allows the coexpression of gene products by a message from a single promoter.

Alternatively, in some cases, a single promoter can direct the expression of an RNA that contains, in a single open reading frame (ORF), two or three polypeptides separated from each other by sequences encoding an autocleaving peptide (for example, 2A sequences) or a protease recognition site (eg, furin), as in the present document described.

The ORF thus encodes a single polypeptide, which, during (in the case of 2A) or after translation, is processed into the individual proteins.

In some embodiments, the "tandem cassette" includes the first component of the cassette which comprises a sequence without a promoter, followed by a transcription termination sequence, and a second sequence, which encodes an autonomous expression cassette or an expression sequence multicistronic. In some embodiments, the tandem cassette encodes two or more polypeptides or different factors, for example, two or more chains or domains of a recombinant receptor. In some embodiments, the nucleic acid sequences encoding two or more chains or domains of the recombinant receptor are introduced as tandem expression cassettes or bi or multicistronic cassettes, at a target DNA integration site.

[00511] The transgene can be inserted into an endogenous gene so that all, some or none of the endogenous gene is expressed. In some embodiments, the transgene (for example, with or without peptide coding sequences) is integrated into any endogenous locus. In some embodiments, the transgene is integrated into the TRAC, TRBC1 and / or TRBC2 gene loci.

[00512] In some embodiments, exogenous sequences may also include transcriptional or translational regulatory sequences, for example, promoters, enhancers, isolators, internal ribosome entry sites, sequences encoding 2A peptides and / or polyadenylation. In addition, the control elements of the genes of interest can be operatively linked to reporter genes to create chimeric genes (for example, reporter expression cassettes). In addition, binding receptor sequences can be included. Exemplary known binding receptor site sequences include, for example, CTGACCTCTTCTCTT-CCTCCCACAG, (SEQ ID NO: 119) (from the human HBB gene) and TTT-CTCTCCACAG (SEQ ID NO: 120) (from the Immunoglobulin-gamma gene) human).

[00513] In an exemplary embodiment, the standard polynucleotide includes homology arms for targeting the TRAC locus, regulatory sequences, for example, promoter, and acid sequences

of the nucleic encoding a recombinant receptor, for example, TCR. In an exemplary embodiment, an additional standard polynucleotide is used, which includes homology arms for targeting TRBC1 and / or TRBC2 loci, regulatory sequences, for example, promoter, and nucleic acid sequences encoding another factor.

[00514] In some embodiments, exemplary standard polynucleotides contain a transgene encoding a recombinant T cell receptor under the operable control of the human elongation factor 1 alpha (EF1α) promoter with HTLV1 enhancer (sequence shown in SEQ ID NO : 127) or the MND promoter (sequence shown in SEQ ID NO: 126) or linked to nucleic acid sequences encoding a P2A ribosome bounce element (sequence shown in SEQ ID NO: 8) to drive expression of the Recombinant TCR from the endogenous target gene locus (eg, TRAC), approximately 800 bp 5 'homology arm sequence (for example, shown in SEQ ID NO: 124), homo arm sequence - 3 'logy of approximately 800 bp (for example, shown in SEQ ID NO: 125) which are homologous to the sequences surrounding the target integration site in exon 1 of the human α TCR constant region (TRAC) gene. In some embodiments, the standard polynucleotide contains yet other nucleic acid sequences, for example, nucleic acid sequences that encode a marker, for example, a surface marker or a selection marker. In some embodiments, the standard polynucleotide also contains viral vector sequences, for example, adenoassociated virus vector (AAV) sequences.

[00515] The transgene contained in the standard polynucleotide described in this document can be isolated from plasmids, cells or other sources using conventional techniques known as PCR. The standard polynucleotide for use can include various types of topology,

including supercoiled circular, relaxed circular, linear and the like. Alternatively, they can be chemically synthesized using standard oligonucleotide synthesis techniques. In addition, standard polynucleotides can be methylated or non-methylated. Standard polynucleotides can be in the form of artificial bacterial or yeast chromosomes (BACs or YACs).

[00516] A polynucleotide can be introduced into a cell as part of a vector molecule having additional sequences such as origins of replication, promoters and genes encoding antibiotic resistance. In addition, standard polynucleotides can be introduced as bare nucleic acid, as nucleic acid complexed with materials such as a liposome, nanoparticle or poloxamer, or can be released by viruses (eg, adenoviruses, AAV , herpesvirus, retrovirus, lentivirus and defective lentivirus integrase (IDLV)).

[00517] In other respects, the standard polynucleotide is released by viral and / or non-viral gene transfer methods. In preferred modes, the standard polynucleotide is released into the cell via an adenoassociated virus (AAV). Any AAV vector can be used, including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 and combinations thereof. In some examples, AAV comprises LTRs that are of a heterologous serotype compared to the capsid serotype (for example, AAV2 ITRs with AAV5, AAV6, or AAV8 capsids). The standard polynucleotide can be released using the same gene transfer system when used to release the nuclease (including in the same vector) or it can be released using a different release system that is used for the nuclease. In some embodiments, the standard polynucleotide is released using a viral vector (for example, AAV) and the nuclease (s) is (are) released in the form of mRNA. The cell

it can also be treated with one or more molecules that inhibit the binding of the viral vector to a cell surface receptor, as in the present document described before, simultaneously and / or after the release of the viral vector (for example, loading the standard nuclease (s) and / or polynucleotide).

[00518] In some embodiments, the standard polynucleotide is comprised of a viral vector, and is at least about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4,250, 4500, 4760, 5000, 5250 , 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10,000 nucleotides in length, or any value between any of the above. In some embodiments, the polynucleotide is comprised of a viral vector, and is between or about 2500 and or or about 5,000 nucleotides, of or about 3,500 and of or about 4,500 nucleotides, or of or about 3,750 nucleotides and of or about 4,250 nucleotides in length. In some embodiments, the polynucleotide is comprised of a viral vector, and is about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4,250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000 , 7500, 8000, 9000 or 10,000 nucleotides in length.

[00519] In some embodiments, the standard polynucleotide is an adenovirus vector, for example, an AAV vector, for example, an ssDNA molecule of a length and sequence that allows it to be packaged in an AAV capsid. The vector may, for example, be less than 5 kb and may contain an ITR sequence that promotes packaging in the capsid. The vector may be deficient in integration. In some embodiments, the standard polynucleotide comprises about 150 to 1000 nucleotides of homology on each side of the transgene and / or the target site. In some embodiments, the standard polynucleotide comprises about 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5 'from the target or transgene site, 3' from target or transgene site, or both 5 'and 3' to the target or transgene site. In some embodiments, the standard polynucleotide comprises at least 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5 'from the target site or transgene, 3' from the target site or transgene, or both 5 'and 3' from the target site or transgene. In some embodiments, the standard polynucleotide comprises a maximum of 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides 5 'from the target site or transgene, 3' from the target site or transgene, or both 5 'and 3' from the target site or transgene.

[00520] In some embodiments, the standard polynucleotide is a lentiviral vector, for example, an IDLV (integration deficiency lentivirus). In some embodiments, the standard polynucleotide comprises about 500 to 1000 base pairs of homology on each side of the transgene and / or the target site. In some embodiments, the standard polynucleotide comprises about 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2,000 base pairs of 5 'homology of the target or transgene site, 3' of the target or transgene site, or both 5 'and 3' to the target or transgene site. In some embodiments, the standard polynucleotide comprises at least 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2,000 base pairs of 5 'of the target site or transgene, 3' of the target site or transgene, or both 5 'and 3' from the target site or transgene. In some embodiments, the standard polynucleotide comprises no more than 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 base pairs of 5 'homology of the target or transgenic site, 3 'from the target or transgene site, or both 5' and 3 'from the target or transgene site. In some embodiments, the standard polynucleotide comprises one or more mutations, for example, silent mutations that prevent Cas9 from recognizing and cleaving the standard polynucleotide. The standard polynucleotide can comprise, for example, at least 1, 2, 3, 4, 5, 10, 20, or 30 silent mutations with respect to the corresponding sequence in the genome of the cell to be altered. In some embodiments, the standard polynucleotide comprises at most 2, 3, 4, 5, 10, 20, 30, or 50 silent mutations in relation to the corresponding sequence in the genome of the cell to be altered.

[00521] The standard double-stranded polynucleotides in the present document described may include one or more bases and / or unnatural backbones. In particular, insertion of a standard polynucleotide with methylated cytosines can be performed using the methods described herein to achieve a state of transcriptional quiescence in a region of interest.

[00522] The standard polynucleotide can comprise any transgene of interest (exogenous sequence). Exemplary exogenous sequences include, however, are not limited to any polypeptide coding sequence (for example, cDNAs or fragments thereof), promoter sequences, enhancer sequences, epitope tags, marker genes, cleavage enzyme recognition sites and various types of expression constructions. The marker genes include, however, are not limited to, sequences encoding proteins that mediate antibiotic resistance (for example, ampicillin resistance, neomycin resistance, G418 resistance, puromycin resistance), sequences that encode colored or fluorescent proteins or luminescent (for example, green fluorescent protein, enhanced green fluorescent protein, red fluorescent protein, luciferase), proteins that measure increased cell growth and / or amplification of the gene (for example, dihydrofolate reductase). Epitope markers include, for example, one or more copies of FLAG, His, myc, Tap, HA or any detectable amino acid sequence.

[00523] In some embodiments, the transgene comprises a polynucleotide that encodes any polypeptide whose expression in the cell is desired, including, but not limited to, antigens,

zymes, receptors (cell or nuclear surface), hormones, lymphokines, cytokines, reporter polypeptides, growth factors and functional fragments of any of the above. In some embodiments, the exogenous sequence (transgene) comprises a polynucleotide that encodes one or more recombinant receptor (s), for example, functional non-TCR antigen receptors, chimeric antigen receptors (CARs), and receptors for T cells (TCRs), such as transgenic TCRs, modified TCRs or recombinant TCRs, and components of any of the above.

[00524] In some embodiments, the coding sequences can be, for example, cDNAs. The exogenous sequences can also be a fragment of a transgene for binding to an endogenous gene sequence of interest. For example, a fragment of a transgene sequence comprising at the 3 'end of a gene of interest can be used to correct, by insertion or replacement, a sequence encoding a mutation at the 3' end of a gene sequence endogenous. Likewise, the fragment may comprise sequences similar to the 5 'end of the endogenous gene for insertion / replacement of endogenous sequences to correct or modify such endogenous sequence. In addition, the fragment can encode a functional domain of interest (catalytic, secretory or similar) for in situ binding to an endogenous gene sequence to produce a fusion protein.

[00525] In some embodiments, the transgene further encodes one or more marker (s). In some embodiments, the one or more marker (s) is (are) a transduction marker, substitute marker and / or a selection marker.

[00526] In some embodiments, the marker is a transduction marker or a substitute marker. A transduction marker or a substitute marker can be used to detect cells that have been introduced with the polynucleotide, for example, a polynucleotide that encodes a recombinant receptor. In some modalities, the transduction marker can indicate or confirm the modification of a cell. In some embodiments, the substitute marker is a protein that is made to be coexpressed on the cell surface with the recombinant receptor, for example, TCR or CAR. In particular embodiments, this substitute marker is a surface protein that has been modified to have little or no activity. In certain embodiments, the substitute marker is encoded in the same polynucleotide that encodes the recombinant receptor. In some embodiments, the nucleic acid sequence that encodes a recombinant receptor is operably linked to a nucleic acid sequence that encodes a marker, optionally separated by an internal ribosome entry site (IRES), or a nucleic acid that encodes a self-cleaving peptide or a peptide that causes the ribosome to bounce, such as a 2A sequence, such as a T2A, a P2A, an E2A or an F2A. Extrinsic marker genes can, in some cases, be used in connection with modified cells to allow the detection or selection of cells and, in some cases, also to promote cellular suicide.

[00527] Exemplary substitute markers may include truncated forms of cell surface polypeptides, such as truncated forms that are non-functional and do not transduce or are not able to transduce a signal or a signal normally transduced by the full length form of the polypeptide of the cell surface, and / or are unable to internalize. Exemplary truncated cell surface polypeptides including truncated forms of growth factors or other receptors such as a truncated human epidermal growth factor 2 (tHER2) receptor, a truncated epidermal growth factor receptor (tEGFR, sequence of tEGFR example

example mentioned in SEQ ID NO: 12 or 13) or a prostate-specific membrane antigen (PSMA) or in a modified form thereof. TEGFR can measure an epitope recognized by the cetuximab antibody (Erbitux®) or another therapeutic anti-EGFR antibody or binding molecule, which can be used to identify or select cells that have been designed with the tEGFR construct and a coded exogenous protein , and / or eliminate separate cells that express the encoded exogenous protein. See U.S. Patent No. 8,802,374 and Liu et al., Nature Biotech. April 2016; 34 (4): 430–434). In some respects, the marker, for example, substitute marker, includes all or part (for example, truncated form) of CD34, an NGFR, a CD19 or a truncated CD19, for example, a truncated non-human CD19, or receptor epidermal growth factor (for example, tEGFR). In some embodiments, the marker is or comprises a fluorescent protein, such as green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), such as super-duplicating GFP (sfGFP), red fluorescent protein (RFP), such as tdTomato, mCherry, mStrawberry, AsRed2, DsRed or DsRed2, cyan fluorescent protein (CFP), bluish green fluorescent protein (BFP), enhanced blue fluorescent protein (EBFP), yellow fluorescent protein (YFP), and variants of them, including species variants, monomeric variants, and codon-optimized and / or enhanced variants of fluorescent proteins. In some embodiments, the marker is or comprises an enzyme, such as a luciferase, the E. coli lacZ gene, alkaline phosphatase, secreted embryonic alkaline phosphatase (SEAP), chloramphenicol acetyltransferase (CAT). Exemplary light-emitting reporter genes include luciferase (luc), β-galactosidase, chloramphenicol acetyltransferase (CAT), β-glucuronidase (GUS) or variants thereof.

[00528] In some modalities, the marker is a selection marker. In some modalities, the selection marker is or comprises a polypeptide that confers resistance to exogenous agents or drugs. In some modalities, the selection marker is an antibiotic resistance gene. In some embodiments, the selection marker is an antibiotic resistance gene that imparts antibiotic resistance to a mammalian cell. In some modalities, the selection marker is or comprises a puromycin resistance gene, a hygromycin resistance gene, a Blasticidin resistance gene, a Neomycin resistance gene, a Geneticin resistance gene or a Zeocin resistance gene or a modified form thereof.

[00529] In some embodiments, the nucleic acid encoding a marker is operably linked to a polynucleotide that encodes a ligand sequence, such as a clickable ligand sequence, for example, a T2A. For example, a marker, and optionally a linker sequence, can be any as described in PCT Pub. No. WO 2014031687. For example, the marker can be a truncated EGFR (tEGFR) which is optionally linked to a linker sequence, such as a T2A cleavable linker sequence. An exemplary polypeptide for a truncated EGFR (for example, tEGFR) comprises an amino acid sequence shown in SEQ ID NO: 12 or 13 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 12 or 13.

[00530] In some embodiments, the marker is a molecule, for example, cell surface protein, not found naturally in cells or not found naturally on the surface of cells, or a portion of them.

[00531] In some modalities, the molecule is a non-own molecule, for example, non-own protein, that is, one that is not recognized as "own" by the host's immune system in which the cells will be adopted transferred.

[00532] In some modalities, the marker has no therapeutic function and / or does not produce any effects besides being used as a marker for genetic engineering, for example, to select successfully modified cells. In other embodiments, the marker may be a therapeutic molecule or a molecule otherwise having some desired effect, such as a ligand for a cell to be found in vivo, such as an immune checkpoint or co-stimulatory molecule to enhance and / or decrease cell responses after adoptive transfer and encounter with the ligand.

[00533] In some embodiments, such a transgene further includes a T2A ribosomal hop element and / or a sequence encoding a marker such as a tEGFR sequence, for example, downstream of a sequence encoding a TCR chain, as shown in SEQ ID NO: 12 or 13, respectively, or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93 %, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity for SEQ ID NO: 12 or 13.

[00534] In some embodiments, the standard polynucleotide encodes a recombinant receptor that serves to direct the function of a T cell. Examples of such encoded recombinant receptors include recombinant T cell receptors (TCRs). In some cases, a chimeric antigen receptor (CAR) is encoded. Chimeric antigen receptors (CARs) are molecules designed to direct immune system cells to specific molecular targets expressed on cell surfaces. In their most basic form, they are receptors introduced into a cell that couple a specificity domain expressed outside the cell with signaling pathways inside the cell, so that when the specificity domain interacts with its target, the cell becomes activated. Often, CARs are made up of variants of T cell receptors (TCRs), where a specificity domain, such as an scFv or some type of receptor, is fused to the signaling domain of a TCR. These constructs are then introduced into a T cell allowing the T cell to become activated in the presence of a cell that expresses the target antigen, resulting in the attack on the target cell by the activated T cell in a non-MHC dependent manner ( see, Chi- caybam et al (2011) Int Rev Immunol 30: 294-311). Alternatively, the CAR expression cassettes can be introduced into an immune cell for posterior grafting so that the CAR cassette is under the control of a specific T cell promoter (for example, the FOXP3 promoter, see Mantel et. Al ( 2006) J. Immunol 176: 3593-3602).

[00535] In an exemplary embodiment, the standard polynucleotide is included as an adeno-associated virus vector (AAV) construct, which contains a nucleic acid sequence encoding recombinant TCR α and TCR β chains under the control of a promoter constitutive, flanked by 800 base pair homology arms each on the 5 'and 3' side of the nucleic acid sequence encoding the recombinant TCR for targeting exon 1 of the endogenous TRAC gene. Exemplary 5 'homology arm for targeting in TRAC includes the sequence shown in SEQ ID NO: 124. Exemplary 3' homology arm for targeting in TRAC includes the sequence shown in SEQ ID NO: 125.

[00536] The construct of such expression cassettes, following the teachings of the present specification, uses methodologies that are known in molecular biology (see, for example, Ausubel or Manniatis). Prior to using the expression cassette to generate a transgenic animal, the expression cassette's responsiveness to the stress inducer associated with selected control elements can be tested by introducing the expression cassette into a suitable cell line (for example, cells primary cells, transformed cells, or immortalized cell lines).

[00537] Targeted insertion of the non-coding nucleic acid sequence can also be achieved. The sequences encoding antisense RNAs, RNAi, shRNAs and micro RNAs (miRNAs) can also be used for targeted insertions. In additional modalities, the standard polynucleotide may comprise non-coding sequences that are specific target sites for additional nuclease projects. Subsequently, additional nucleases can be expressed in cells so that the original standard polynucleotide is cleaved and modified by the insertion of another standard polynucleotide of interest. In this way, repetitive integrations of standard polynucleotides can be generated allowing the stacking of characteristics in a locus of particular interest, for example, TRAC, TRBC1 and / or TRBC2 gene loci.

[00538] In some embodiments, the polynucleotide contains the structure: [5 'homology arm] - [transgene sequence] - [3' homology arm]. In some embodiments, the polynucleotide contains the structure: [5 'homology arm] - [multicistronic element] - [transgene sequence] - [3' homology arm]. In some modalities, the polynucleotide contains the structure: [5 'homology arm] - [promoter] - [transgene sequence] - [3' homology arm].

4. Release of Standard Polynucleotides

[00539] In some embodiments, the polynucleotide, for example, a polynucleotide such as a standard polynucleotide encoding a chimeric receptor, is introduced into cells in the form of a nucleotide, for example, as a polynucleotide or a vector. In modali-

particular activities, the polynucleotide contains a transgene that encodes the chimeric receptor or a portion of it.

[00540] In some embodiments, the polynucleotide, for example, the standard polynucleotide, is introduced into the cell to modify, in addition to agents capable of introducing a target genetic disruption, for example, nuclease and / or gRNAs. In some embodiments, standard polynucleotides can be released before, simultaneously, or after agents capable of introducing a target genetic disruption are introduced into a cell. In some embodiments, standard polynucleotides are released simultaneously with the agents. In some embodiments, standard polynucleotides are released before agents, for example, seconds to hours to days before agents, including, but not limited to, 1 to 60 minutes (or any time between them), before agents, from 1 to 24 hours (or any time between), before agents or more than 24 hours before agents. In some embodiments, standard polynucleotides are released after the agents, seconds to hours to days after the agents, including immediately after the agent is released, for example, between 1 minute to 4 hours, such as about 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after release of agents and / or preferably within 4 hours of release of agents. In some embodiments, the standard polynucleotide is released more than 4 hours after the agents are released. In some embodiments, standard polynucleotides are released after agents, for example, including, but not limited to, within 1 second to 60 minutes (or any time between them) after agents, 1 to 4 hours (or any time between) after the agents or more than 4 hours after the agents.

[00541] In some embodiments, standard polynucleotides may

they can be released using the same delivery systems as the agent (s) capable of introducing a target genetic disruption, for example, nuclease and / or gRNAs. In some embodiments, standard polynucleotides can be released using different delivery systems, the same as the agent (s) capable of introducing a target genetic disruption, for example, nuclease and / or gRNAs. In some embodiments, the standard polynucleotide is released simultaneously with the agent (s). In other modalities, the standard polynucleotide is released at a different time, before or after the release of the agent (s). Any of the release methods described in this document, for example, in Section IA3 as in Tables 6 and 7, for releasing nucleic acids in the agent (s) capable of introducing a target genetic disruption, for example For example, nuclease and / or gRNAs, can be used to release the standard polynucleotide.

[00542] In some embodiments, said one or more agents and the standard polynucleotide are released in the same format or method. For example, in some embodiments, said one or more agents and the standard polynucleotide are also compressed into a vector, for example, a viral vector. In some embodiments, the standard polynucleotide is encoded in the same vector backbone, for example, AAV genome, plasmid DNA, such as Cas9 and gRNA. In some respects, said one or more agents and the standard polynucleotide are in different formats, for example, ribonucleic acid-protein complex (RNP) for the Cas9-gRNA agent and a linear DNA for the polynucleotide default, but they are released using the same method. In some respects, said one or more agents and the standard polynucleotide are in different formats, for example, ribonucleic acid-protein complex (RNP) for the Cas9-gRNA agent and the standard polynucleotide is contained in an AAV vector, and the RNP is released using a physical release method

(for example, electroporation) and the standard polynucleotide is released by transducing viral AAV preparations. In some aspects, the standard polynucleotide is released immediately after, for example, within about 1, 2, 3, 4, 5, 10, 20, 30, 40, 50 or 60 minutes after the release of the said one or more agents.

[00543] In some embodiments, the standard polynucleotide is a linear or circular nucleic acid molecule, such as a linear RNA or linear or circular DNA, and can be released using any of the methods described in Section IA3 herein. (for example, Tables 6 and 7) for releasing nucleic acid molecules into the cell.

[00544] In particular modalities, the polynucleotide, for example, the standard polynucleotide, is introduced into cells in the form of nucleotides, for example, as or within a non-viral vector. In some embodiments, the non-viral vector is or includes a polynucleotide, for example, a DNA or RNA polynucleotide, which is suitable for transduction and / or transfection by any suitable and / or known non-viral method for gene release, such as, but not limited to, microinjection, electroporation, transient cell compression or squeezing (for example, as described in Lee, and other (2012) Nano Lett 12: 6322-27), lipid-mediated transfection , peptide-mediated release, for example, cell penetrating peptides, or a combination thereof. In some embodiments, the non-viral polynucleotide is released into the cell by a non-viral method described herein, such as a non-viral method listed in Table 7 herein.

[00545] In some embodiments, the standard polynucleotide sequence can be compressed into a vector molecule that contains sequences that are not homologous to the region of interest in the genetic DNA. In some embodiments, the virus is a DNA virus (for example, dsDNA or ssDNA virus). In some embodiments, the virus is an RNA virus (for example, ssRNA or dsRNA virus). Exemplary viral / virus vectors include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, poxviruses, and simple herpes viruses, or any of the viruses described elsewhere. place in this document.

[00546] In some embodiments, the standard polynucleotide can be transferred into cells using recombinant infectious virus particles, such as, for example, vectors derived from simian virus 40 (SV40), adenovirus, adeno-associated virus (AAV) ). In some embodiments, the standard polynucleotide is transferred to T cells using recombinant lentiviral vectors or retroviral vectors, such as gamma-retroviral vectors (see, for example, Koste and others (2014) Gene Therapy, April 3, 2014. doi: 10.1038 / gt.2014.25; Carlens and other (2000) Exp Hematol 28 (10): 1137-46; Alonso- Camino and other (2013) Mol Ther Nucl Acids 2, e93; Park and other (s), Trends Biotechnol. November 29, 2011 (11): 550–557 or HIV-1-derived lentiviral vectors.

[00547] In some modalities, the retroviral vector has a long terminal repeat (LTR) sequence, for example, a retroviral vector derived from the murine leukemia virus Moloney (MoMLV), myeloproliferative sarcoma virus (MPSV), murine embryonic stem cell virus (MESV), murine stem cell virus (MSCV), virus forming a spleen focus (SFFV). Most retroviral vectors are derived from murine retroviruses. In some embodiments, retroviruses include those derived from any bird or mammal cell source. Retroviruses are typically amphoteric, meaning that they are capable of infecting host cells of various species, including humans. In one embodiment, the gene to be expressed replaces the retroviral sequences gag, pol, and / or env. Several illustrative retroviral systems have been described (for example, U.S. Patent Nos. 5,219,740; 6,207,453; 5,219,740; Miller and Rosman (1989) BioTechniques 7: 980-990; Miller, AD (1990) Human Gene Therapy 1 : 5-14; Scarpa and others (1991) Virology 180: 849-852; Burns and others (1993) Proc. Natl. Acad. Sci. USA 90: 8033-8037; and Boris-Lawrie and Temin (1993) Cur. Opin. Genet. Develop. 3: 102-109).

[00548] In some embodiments, the standard polynucleotides and nucleases can be in the same vector, for example, an AAV vector (for example, AAV6). In some embodiments, standard polynucleotides are released using an AAV vector the agent (s) capable of introducing a target genetic disruption, for example, nuclease and / or gRNAs are released as a different form, for example , as mRNAs that encode the nucleases and / or gRNAs. In some modalities, standard polynucleotides and nucleases are released using the same type of method, for example, a viral vector, but in separate vectors. In some embodiments, standard polynucleotides are released in a different delivery system like agents capable of introducing a genetic disruption, for example, nucleases and / or gRNAs. In some embodiments, the standard polynucleotide is excised from a backbone of an in vivo vector, for example, it is flanked by gRNA recognition sequences. In some embodiments, the standard polynucleotide is in a separate polynucleotide molecule such as Cas9 and gRNA. In some embodiments, Cas9 and gRNA are introduced as a ribonucleoprotein complex (RNP), and the standard polynucleotide is introduced as a polynucleotide molecule, for example, in a vector or linear nucleic acid molecule, for example, DNA linear. Types or nucleic acids and vectors for release include any of those in Section III herein.

5. Genetically modified locus

[00549] In some embodiments, the methods, compositions, articles of manufacture, and / or kits provided herein are useful for generating, manufacturing, or producing genetically modified cells, for example, genetically modified immune cells and / or T cells, which have or contain a modified gene locus. In particular embodiments, the methods provided in this document result in genetically modified cells that have or contain a modified gene locus. In particular embodiments, the modified locus is or contains a fusion of a transgene, for example, a transgene described in Section I.B, and an open reading frame for an endogenous gene. In certain embodiments, the transgene encodes a part of a recombinant protein and is inserted into a frame in an open reading frame of the endogenous gene, resulting in a modified locus that encodes all recombinant protein. In some embodiments, the recombinant protein is a recombinant receptor. In some embodiments, the recombinant protein is a recombinant TCR.

[00550] In certain embodiments, the transgene encodes a part of a recombinant TCR and is inserted within an endogenous open reading structure that encodes a constant domain of TCR. In some embodiments, the modified locus encodes the complete recombinant TCR. In particular embodiments, a portion of the encoded recombinant TCR is encoded by a nucleic acid sequence present in the transgene, and the remaining portion of the recombinant TCR is encoded by a nucleic acid sequence present in an open reading frame of the gene endogenous. In particular modes, transcription of the modified locus results in an mRNA encoding the recombinant TCR. In particular embodiments, a portion of the mRNA is transcribed from a nucleic acid sequence present in the transgene, and the remaining portion of the mRNA is transcribed from a nucleic acid sequence present in an open reading frame of the endogenous gene . In some embodiments, the transgene is integrated into a target site immediately upstream of and in the structure with, the region or part of the open reading frame that encodes the remaining portion of the recombinant TCR.

[00551] In some embodiments, the mRNA transcribed from the modified locus contains a 3'UTR that is encoded by the endogenous gene and / or is identical to a 3'UTR of an mRNA that is transcribed from the endogenous gene, for example , endogenous and / or unmodified TRBC or TRAC gene. In some embodiments, the transgene contains a ribosomal jump element upstream, for example, immediately upstream, of a nucleic acid sequence that encodes a part of the TCR. In certain embodiments, transcription of the modified locus results in an mRNA encoding the recombinant TCR. In some modalities, the mRNA encoding the recombinant TCR contains a 5'UTR that is encoded by the endogenous gene and / or is identical to a 5'UTR of an mRNA that is transcribed from the endogenous gene, for example, a TRBC gene or endogenous and / or unmodified TRAC.

[00552] In certain embodiments, the modified locus contains a nucleic acid sequence that encodes a recombinant TCR having fewer introns than a locus, for example, an endogenous or unmodified locus, that encodes an endogenous and / or native TCR. In particular modes, a nucleic acid sequence encoding the recombinant TCR has one, two, three, four, five, six, seven, eight, nine, ten or more than ten less introns than a nucleic acid sequence from or within an unmodified locus, for example, an endogenous locus, which encodes a native and / or endogenous TCR. In certain embodiments, the nucleic acid sequence encoding the recombinant TCR has fewer introns than an unmodified TRAC locus. In particular embodiments, the nucleic acid sequence encoding the recombinant TCR has fewer introns than an unmodified TRBC locus, for example, TRBC1 and / or TRBC2.

[00553] In certain embodiments, the modified locus contains a nucleic acid sequence that encodes a recombinant TCR, and the nucleic acid sequence contains at least one, at least two, at least three, at least four, at least five, at least at least six, at least seven, at least eight, at least nine, or at least ten introns. In particular embodiments, the nucleic acid sequence contains an intron. In particular embodiments, the nucleic acid sequence contains two introns. In certain embodiments, the nucleic acid sequence contains three introns. In some modalities, the introns are not within the nucleic acid sequence of the transgene.

[00554] In some embodiments, the modified locus is a modified TRAC locus. In some embodiments, the modified TRAC locus is or contains a fusion of a transgene and an open reading frame of the endogenous TRAC locus. In some embodiments, the modified locus encodes a recombinant TCR receptor of natural, complete and / or total length, a portion of which is encoded by the transgene, for example, by a nucleic acid sequence from or within the transgene, and the remaining portion of which is encoded by the open reading frame of the endogenous TRAC gene, for example, a nucleic acid sequence in or within the open reading frame of the endogenous TRAC gene. In some embodiments, the transgen encodes part of a TCRα chain and the open reading frame encodes the remaining portion of a TCRα chain. In certain embodiments, the transgene encodes a TCRβ chain and part of a TCRα chain and the open reading frame encodes the remaining portion of a TCRα chain. In some embodiments, the transgene encodes a variable domain of a TCRα chain and the open reading frame encodes the constant region of a TCRα chain. In certain embodiments, the transgene encodes a variable domain of a TCRα chain and a part of the constant domain of a TCRα chain and the open reading frame encodes the remaining portion of the constant domain of TCRα.

[00555] In some embodiments, the modified locus is a modified TRBC locus, for example, TRBC1 or TRBC2. In some modalities, the modified TRBC locus is or contains a fusion of a transgene and an open reading frame of the endogenous TRBC locus. In some embodiments, the modified locus encodes a recombinant TCR receptor of natural, full and / or full length, a portion of which is encoded by the transgene, for example, by a nucleic acid sequence from or within the transgene, and the remaining portion of which is encoded by the open reading frame of the endogenous TRBC gene, for example, a nucleic acid sequence in or within the open reading frame of the endogenous TRAC gene. In some embodiments, the transgene encodes part of a chain of TCRβ and the open reading frame encodes the remainder of the TCRβ chain. In certain embodiments, the transgene encodes a TCRα chain and part of a TCRβ chain and the open reading frame encodes the remaining part of the TCRβ chain. In some embodiments, the transgene encodes the variable domain of a TCRβ chain and the open reading frame encodes the constant region of the TCRβ domain. In certain embodiments, the transgene co-replicates a variable domain of the TCRβ and a part of the constant domain of a TCRβ chain and the open reading frame encodes the remaining portion of the constant domain of TCRβ.

[00556] In some embodiments, a recombinant TCR encoded by the modified locus is a functional TCR. In some modalities

des, a recombinant TCR encoded by the modified locus binds to and / or is able to bind to a target antigen. In some modalities, the target antigen is associated with, specific to, and / or expressed in a cell or tissue that is associated with a disease, disorder or condition. In some embodiments, the disease, disorder or condition is or includes an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer. In some embodiments, the target antigen is a tumor antigen or a pathogenic antigen. In particular modalities, the pathogenic antigen is a bacterial or viral antigen. In certain modalities, a TCRα chain that is encoded by the modified TRAC locus binds and / or is able to bind to a TCRβ chain. In some embodiments, a TCRβ chain that is encoded by the modified TRBC locus, for example, a TRBC1 or TRBC2 locus, is able to bind to a TCRα locus. II. Nucleic acids, vectors and release

[00557] In some embodiments, the polynucleotide, for example, a polynucleotide such as a standard polynucleotide that encodes a recombinant receptor and / or TCR, is introduced into cells in the form of a nucleotide, for example, as a polynucleotide or vector. In particular embodiments, the polynucleotide contains a transgene that encodes the recombinant receptor and / or TCR. In certain embodiments, said one or more agents for genetic disruption are introduced into cells in the form of nucleic acid, for example, as polynucleotides and / or vectors. In some embodiments, the components for modification can be released in various forms using various release methods, including the components encoding the polynucleotides, for example, as described in Section I. Also provided are one or more polynucleotides (for example, example, nucleic acid molecules) encoding one or more components of said one or more agents capable of inducing a genetic disruption, and / or one or more standard polynucleotides containing transgene, and vectors for genetically modified cells for targeted integration of the transgene.

[00558] In some embodiments, polynucleotides are provided, for example, standard polynucleotides for targeting transgene at a specific genomic target location, for example, at the TRAC, TRBC1 and / or TRBC2 locus. In some modalities, provided are any standard polynucleotides described in this document, for example, in Section IB In some modalities, the standard polynucleotide contains transgene that includes nucleic acid sequences that encode a recombinant receptor or other polypeptides and / or factors, and homology arms for targeted integration. In some embodiments, the standard polynucleotide can be contained in a vector.

[00559] In some modalities, agents capable of introducing a genetic disruption can be encoded in one or more polynucleotides. In some embodiments, the component of the agents, for example, Cas9 molecule and / or a gRNA molecule, can be encoded in one or more polynucleotides, and introduced into cells. In some embodiments, the polynucleotide that encodes one or more components of the agents can be included in a vector.

[00560] In some embodiments, a vector may comprise a sequence encoding a Cas9 molecule and / or a standard gRNA and / or polynucleotide molecule. A vector may also comprise a sequence encoding a signal peptide (for example, for nuclear, nucleolar, mitochondrial location), fused, for example, to a sequence of Cas9 molecule. For example, a vector can comprise a nuclear localization sequence (for example, from SV40) fused to a sequence that encodes a Cas9 molecule.

[00561] In particular modalities, one or more regulatory / control elements, for example, a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, internal ribosome entry sites (IRES), a 2A string, and splice donor or acceptor can be included in the vectors. In some modalities, the promoter is selected from a pol I, pol II or pol III RNA promoter. In some embodiments, the promoter is known as RNA polymerase II (for example, a major adenovirus late promoter or early SV40 region, CMV). In another embodiment, the promoter is recognized by RNA polymerase III (for example, a U6 or H1 promoter).

[00562] In certain embodiments, the promoter is a regulatory promoter (for example, an inducible promoter). In some embodiments, the promoter is an inducible promoter or a repressible promoter. In some embodiments, the promoter comprises a Lac operator sequence, a tetracycline operator sequence, a galactose operator sequence or a doxycycline operator sequence, or is an analog of the same or is capable of being linked by or recognized - acted by a Lac repressor or a teTRACiclina repressor, or an analog thereof.

[00563] In some embodiments, the promoter is or comprises a constitutive promoter. Exemplary constitutive promoters include, for example, simian virus 40 (SV40) early promoter, cytomegalovirus (CMV) early promoter, human Ubiquitin C (UBC) promoter, human elongation factor 1α promoter (EF1α), kinase promoter 1 of mouse phosphoglycerate (PGK), and chicken β-Actin promoter coupled with early CMV enhancer (CAGG). In some modalities, the constitutive promoter is a synthetic or modified promoter. In some embodiments, the promoter is or comprises an MND promoter, a synthetic promoter that contains the U3 region of a modified MoMuLV LTR with myeloproliferative sarcoma virus enhancer (sequence mentioned in SEQ ID NO: 18 or 126; see Challita and other (1995) J. Virol. 69 (2): 748-755). In some embodiments, the promoter is a tissue-specific promoter. In another embodiment, the promoter is a viral promoter. In another way, the promoter is a non-viral promoter. In some modalities, exemplary promoters may include, but are not limited to, human elongation factor 1 alpha promoter (EF1α) (sequence mentioned in SEQ ID NO: 4 or 5) or a modified form thereof ( EF1α promoter with HTLV1 enhancer; sequence mentioned in SEQ ID NO: 127) or MND promoter (sequence mentioned in SEQ ID NO: 18 or 126). In some embodiments, the polynucleotide and / or vector does not include a regulatory element, for example, a promoter.

[00564] In particular embodiments, the polynucleotide, for example, the polynucleotide that encodes a recombinant receptor and / or TCR, is introduced into cells in the form of a nucleotide, for example, as or within a non-viral vector. In some embodiments, the non-viral vector is or includes a polynucleotide, for example, a DNA or RNA polynucleotide, which is suitable for transduction and / or transfection by any suitable and / or known non-viral method for gene release, such as, but not limited to, microinjection, electroporation, squeezing, or transient cell compression (for example, as described in Lee, and other (2012) Nano Lett 12: 6322-27), lipid-mediated transfection, release peptide-mediated, or a combination thereof. In some embodiments, the non-viral polynucleotide is released into the cell by a non-viral method described in this document, such as a non-viral method listed in Table 7.

[00565] In some embodiments, the delivery vector or vehicle is a viral vector (for example, for the generation of recombinant viruses). In some embodiments, the virus is a DNA virus (for example, dsDNA or ssDNA virus). In some embodiments, the virus is an RNA virus (for example, an ssRNA virus). Exemplary viral vectors / viruses include, for example, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses (AAV), vaccinia viruses, poxviruses, and herpes simplex viruses, or any virus described elsewhere in this document.

[00566] In some modalities, the virus infects cells in division. In another embodiment, the virus infects non-dividing cells. In another embodiment, the virus infects both divisible and non-divisible cells. In another embodiment, the virus can integrate into the host's genome. In another embodiment, the virus is modified to have reduced immunity, for example, in humans. In another embodiment, the virus is replication-competent. In another embodiment, the virus is deficient for replication, for example, having one or more coding regions for the genes necessary for the additional cycles of virion replication and / or packaging replaced with other genes or deleted. In another modality, the virus causes transient expression of the Cas9 molecule and / or gRNA molecule for the purposes of transient induction of genetic disruption. In another modality, the virus causes long-term expression, for example, at least 1 week, 2 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanent, of the Cas9 molecule and / or gRNA molecule. The packaging capacity of viruses can vary, for example, from at least about 4 kb to at least about 30 kb, for example, at least about 5 kb, 10 kb, 15 kb, 20 kb, 25 kb, 30 kb, 35 kb, 40 kb, 45 kb, or 50 kb.

[00567] In some embodiments, the polynucleotide containing the standard agent (s) and / or polynucleotide is released by a recombinant retrovirus. In another embodiment, the retrovirus (for example, Moloney murine leukemia virus) comprises a reverse transcriptase, for example, which allows integration into the host genome. In some modalities, the retrovirus is replication-competent. In another embodiment, the retrovirus is replication-defective, for example, having one or more coding regions for the genes needed for the additional cycles of virion replication and packaging replaced with other genes, or deleted.

[00568] In some embodiments, the polynucleotide containing the standard agent (s) and / or polynucleotide is released by a recombinant lentivirus. For example, lentivirus is replication-defective, for example, it does not comprise one or more genes required for viral replication. In some embodiments, the virus is an HIV-derived lentivirus.

[00569] In some embodiments, the polynucleotide containing the standard agent (s) and / or polynucleotide is released by a recombinant adenovirus. In another embodiment, the adenovirus is modified to have reduced immunity in humans.

[00570] In some embodiments, the polynucleotide containing the standard agent (s) and / or polynucleotide is released by a corresponding AAV. In some embodiments, AAV can incorporate its genome into that of a host cell, for example, a target cell as in the present document described. In another modality, AAV is a self-complementing adenoassociated virus (scAAV), for example, a scAAV that packs both ring strands together to form double-stranded DNA. AAV serotypes that can be used in the described methods include AAV1, AAV2, modified AAV2 (for example, changes in Y444F, Y500F, Y730F and / or S662V), AAV3, modified AAV3 (for example, changes in

Y705F, Y731F and / or T492V), AAV4, AAV5, AAV6, modified AAV6 (e.g., modifications in S663V and / or T492V), AAV7, AAV8, AAV 8.2, AAV9, AAV.rh10, AAV.rh10 modified, AAV. rh32 / 33, modified AAV.rh32 / 33, modified AAV.rh43, modified AAV.rh43, AAV.rh64R1, modified AAV.rh64R1, and pseudo-typified AAV, such as AAV2 / 8, AAV2 / 5 and AAV2 / 6 can also be used in the methods described.

[00571] In some embodiments, the polynucleotide containing the standard agent (s) and / or polynucleotide is released by a hybrid virus, for example, a hybrid of one or more of the viruses described in this document.

[00572] A packaging cell is used to form a virus particle that is capable of infecting a target cell. Such a cell includes a 293 cell, which can package adenovirus, and a ψ2 cell or a PA317 cell, which can package the retrovirus. A viral vector used in gene therapy is usually generated by a producer cell line that packs a nucleic acid vector into a viral particle. The vector typically contains the minimum viral sequences required for packaging and subsequent integration into a target or host cell (if applicable), with other viral sequences being replaced by an expression cassette encoding a protein to be expressed, for example, Cas9 . For example, an AAV vector used in gene therapy typically only has repeated inverted terminal (ITR) sequences from the AAV genome which are required for packaging and gene expression in the target or host cell. The missing viral functions are provided in trans by the packaging cell line. Hereafter, the viral DNA is packaged in a cell line, which contains an auxiliary plasmid that encodes other AAV genes, named rep and cap, but lacking the ITR sequences. The cell line is also infected with

novirus as a helper. The helper virus promotes the replication of the AAV vector and the expression of the AAV genes in the helper plasmid. The helper plasmid is not packaged in significant quantities due to a lack of the ITR sequences. Contamination with adenovirus can be reduced by, for example, heat treatment to which adenovirus is more sensitive than AAV.

[00573] In some modalities, the viral vector has the ability to recognize the cell type. For example, the viral vector can be pseudotyped with a different / alternative viral envelope glycoprotein; modified with a cell type-specific receptor (for example, genetic modification of viral envelope glycoproteins to incorporate targeting ligands such as a peptide ligand, a single chain antibody, a growth factor); and / or modified to have a molecular bridge with dual specificities with one end recognizing a viral glycoprotein and the other end recognizing a portion of the target cell surface (for example, ligand receptor, monoclonal antibody, chemical conjugation and avidin-biotin ).

[00574] In some modalities, the viral vector reaches cell-specific expression. For example, a specific tissue promoter can be constructed to restrict the expression of the transgene (Cas9 and gRNA) in just one specific target cell. The specificity of the vector can also be mediated by microRNA-dependent control of transgene expression. In some embodiments, the viral vector has increased efficiency of fusing the viral vector and a target cell membrane. For example, a fusion protein such as competent fusion hemagglutinin (HA) can be incorporated to increase viral uptake in cells. In some embodiments, the viral vector has the capability of nuclear localization. For example, a virus that requires the collapse of the nuclear membrane (during

they are cellular) and, therefore, will not infect a non-divisible cell, it can be altered to incorporate a nuclear localized peptide in the virus matrix protein, thus allowing the transduction of non-proliferating cells. III. CELLS FOR GENETIC MODIFICATION

[00575] In some of the modalities provided, cells for modification are immune cells, such as T cells. Supplied are genetically modified cells or cell populations in which one or more of the cells contain a knockout of one or more endogenous TCR genes and nucleic acids which encodes the recombinant receptor and / or another transgene that are integrated into one or more of the endogenous TCR genes. Also provided are populations or compositions of such cells, compositions that contain such cells and / or enriched by cells that are modified using the methods provided.

[00576] In some embodiments, cells for modification include one or more subgroups of T cells or other cell types, such as total T cell populations, CD4 + cells, CD8 + cells, and subpopulations thereof, such as those defined by function, activation state, maturity, potential for differentiation, expansion, recirculation, location and / or persistence capacities, antigen specificity, type of antigen receptor, presence in a particular organ or compartment, marker or profile of cytokine secretion and / or degree of differentiation. With reference to the individual to be treated, the cells can be allogeneic and / or autologous. Methods include methods without a plate. In some respects, such as for non-shelf technologies, cells are pluripotent and / or multipotent, such as stem cells, such as induced pluripotent stem cells (iPSCs). In some modalities, the methods include isolation cells from the individual, preparing, processing, cultivating and / or modifying them, as in the present document described, and reintroducing them to the same patient, before or after cryopreservation.

[00577] Among the subtypes and subpopulations of T cells and / or CD4 + and / or CD8 + T cells are virgin T cells (TN), effector T cells (TEFF), memory T cells and subtypes thereof, as memory T stem cell (TSCM), central memory T (TCM), effector memory T (TEM), or terminally differentiated memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells, mucosa-associated invariant T cells (MAIT), adaptive and naturally occurring regulatory T cells (Treg), helper T cells, such as TH1 cells, TH2 cells, TH3 cells , TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, alpha / beta T cells, and delta / gamma T cells. In some embodiments, the cell is a regulatory T cell (Treg). In some embodiments, the cell also comprises a recombinant FOXP3 or variant thereof.

[00578] In some embodiments, cells include one or more nucleic acids introduced by means of genetic modification and, thus, express genetically modified products or combinations of such nucleic acids. In some embodiments, nucleic acids are heterologous, that is, normally not present in a cell or sample obtained from the cell, such as that obtained from another organism or cell, which, for example, is not ordinarily found in the cell being modified and / or an organism from which such a cell is derived. In some embodiments, nucleic acids are not naturally occurring, such as a nucleic acid not found in nature, including one that comprises chimeric combinations of nucleic acids that encodes multiple domains of different cell types.

[00579] In some modalities, the preparation of the modified cells includes one or more stages of culture and / or preparation. Modification cells can be isolated from a sample, such as a biological sample, for example, one obtained from or derived from an individual. In some modalities, the individual from whom the cell is isolated is someone having the disease or condition or in need of cell therapy or to whom cell therapy will be administered. The individual, in some modalities, is a human in need of a particular therapeutic intervention, such as adoptive cell therapy for cells being isolated, processed and / or modified.

[00580] Consequently, cells, in some embodiments, are primary cells, for example, primary human cells. The samples include tissue, fluid and other samples taken directly from the individual, as well as samples resulting from one or more processing steps, such as separation, centrifugation, genetic modification (for example, transduction with viral vector), washing and / or cubation. The biological sample can be a sample obtained directly from a biological source or a sample that is processed. Biological samples include, but are not limited to, body fluids, such as blood, plasma, serum, cerebrospinal fluid, senile fluid, urine and sweat, tissue and organ samples, including processed samples derived from them.

[00581] In some respects, the sample from which the cells are derived or isolated is blood or a sample derived from blood, or is derived from an apheresis or leukapheresis product. Exemplary samples include whole blood, peripheral blood mononuclear cells (PBMCs), leukocytes, bone marrow, thymus, tissue biopsy, tumor, leukemia, lymphoma, lymph node, lymphoid tissue associated with intestine, lymphoid tissue associated with mucosa, spleen , other lymphoid tissues, liver, lung, stomach, intestine, colon, kidney, pancreas, breast,

bone, prostate, cervix, testicles, ovaries, amygdala or other organ and / or cells derived from them. Samples include, in the context of cell therapy, for example, adoptive cell therapy, samples from autologous and allogeneic sources.

[00582] In some embodiments, the cells are derived from cell lines, for example, T cell lineage. The cells, in some embodiments, are obtained from a xenogeneic source, for example, from mouse, rat, non-human primate. or pig.

[00583] In some embodiments, cell isolation includes one or more preparation and / or cell separation steps based on non-affinity. In some examples, cells are washed, centrifuged and / or incubated in the presence of one or more reagents, for example, to remove unwanted components, enrich for desired components, lyse or remove cells sensitive to particular reagents. In some examples, cells are separated on the basis of one or more properties, such as density, adherent properties, size, sensitivity and / or resistance to particular components.

[00584] In some examples, an individual's circulating blood cells are obtained, for example, by apheresis or or leukapheresis. The samples, in some respects, contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells and / or platelets, and, in some respects, contain cells other than red blood cells. and platelets.

[00585] In some embodiments, the blood cells collected from the individual are washed, for example, to remove the plasma fraction and place the cells in an appropriate buffer or means for subsequent processing steps. In some modalities, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the washing solution lacks calcium and / or magnesium and / or many or all divalent cations. In some aspects, a washing step is performed in a semi-automatic "flow-through" centrifuge (for example, the Cobe 2991 cell processor, Baxter) according to the manufacturer's instructions. In some respects, a washing step is carried out by tangential flow filtration (TFF) according to the manufacturer's instructions. In some embodiments, cells are resuspended in a variety of biocompatible buffers after washing, such as, for example, Ca ++ / Mg ++ free PBS. In certain embodiments, the components of a blood cell sample are removed and the cells are directly resuspended in culture media.

[00586] In some embodiments, the methods include cell separation methods based on density, such as the preparation of peripheral blood white blood cells by lysing red blood cells and centrifugation using a Percoll or Ficoll gradient.

[00587] In some embodiments, isolation methods include separating different types of cells based on the expression or presence in the cell of one or more specific molecules, such as surface markers, for example, surface proteins in inTRACellular markers, or nucleic acid. In some modalities, any known method for separation based on such markers can be used. In some modalities, separation is separation based on affinity or immunoaffinity. For example, isolation, in some respects, includes the separation of cells and cell populations based on cell expression or level of expression of one or more markers, typically cell surface markers, for example, by incubation with an antibody or binding partner that specifically binds to such markers, generally followed by steps of washing and separating cells having binding to the antibody or binding partner, from those cells not

binding to the antibody or binding partner.

[00588] Such separation steps can be based on positive selection, in which cells having binding to the reagents are retained for another use, and / or negative selection, in which cells having no binding to the antibody or binding partner are retained. In some instances, both fractions are retained for another use. In some aspects, negative selection can be particularly useful where no antibody is available that specifically identifies a cell type in a heterogeneous population, such that separation is best performed based on markers expressed by cells other than the desired population. .

[00589] The separation does not need to result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection of or enrichment for cells of a particular type, such as those expressing a marker, refers to increasing the number or percentage of such cells, but does not need to result in a complete absence of cells not expressing the highlighter. Likewise, negative selection, removal or depletion of cells of a particular type, such as those expressing a marker, refers to decreasing the number or percentage of such cells, but does not need to result in complete removal or all such cells.

[00590] In some examples, multiple rounds of separation steps are performed, where the positively or negatively selected fraction of one step is subjected to another separation step, such as a subsequent positive or negative selection. In some instances, a single separation step may deplete cells by expressing multiple markers simultaneously, such as incubating cells with a plurality of antibodies or binding partners, each specific for a targeted marker for neural selection.

gative. Likewise, multiple cell types can simultaneously be positively selected by incubating cells with a plurality of antibodies or binding partners expressed in various cell types.

[00591] For example, in some respects, specific subpopulations of T cells, such as positive cells or expressing high levels of one or more surface markers, for example, CD28 +, CD62L +, CCR7 +, CD27 + T cells , CD127 +, CD4 +, CD8 +, CD45RA +, and / or CD45RO +, are isolated by positive or negative selection techniques.

[00592] For example, CD3 +, CD28 + T cells can be positively selected using magnetic beads conjugated to anti-CD3 / anti-CD28 (for example, DYNABE- ADS® M-450 CD3 / CD28 T-cell expander ).

[00593] In some modalities, isolation is performed by enrichment for a particular cell population by positive selection, or depletion of a particular cell population by negative selection. In some embodiments, positive or negative selection is performed by incubating cells with one or more antibodies or another binding agent that specifically binds to one or more expressed or expressed surface markers (+ marker) at a relatively low level. higher (high marker) in the positively or negatively selected cells, respectively.

[00594] In some embodiments, T cells are separated from a PBMC sample by negative selection of markers expressed on non-T cells, such as B cells, monocytes, or other white blood cells, such as CD14. In some respects, a CD4 + or CD8 + selection step is used to separate CD4 + helper and CD8 + cytotoxic T cells. Such CD4 + and CD8 + populations can also be classified into subpopulations by positive or negative selection by markers.

expressed or expressed to a relatively greater degree in one or more subpopulations of virgin, memory and / or effector T cells.

[00595] In some modalities, CD8 + cells are also enriched by or depleted of virgin cells, of central memory, effector memory, and / or central memory trunk, as well as by positive or negative selection based on antigens from associated with the respective subpopulations. In some embodiments, enrichment for central memory T cells (TCM) is performed to increase efficacy, such as improving long-term survival, expansion and / or graft after administration, which in some respects is particularly robust in such subpopulations. See Terakura and others (2012) Blood.1: 72–82; Wang and others (2012) J Immunherher. 35 (9): 689-701. In some embodiments, combining CD8 + and CD4 + T cells with TCM-enriched T cells also enhances effectiveness.

[00596] In modalities, memory T cells are present in both CD62L + and CD62L- subgroups of CD8 + peripheral blood lymphocytes. PBMC can be enriched for or depleted of CD62L-CD8 + and / or fractions of CD62L + CD8 +, such as using anti-CD8 and anti-CD62L antibodies.

[00597] In some modalities, enrichment for central memory T cells (TCM) is based on the expression of high and positive surface of CD45RO, CD62L, CCR7, CD28, CD3, and / or CD 127; in some respects, it is based on negative selection for cells expressing or highly expressing CD45RA and / or granzyme B. In some respects, isolation of a CD8 + population enriched for TCM cells is accomplished by depletion of cells pressing CD4, CD14, CD45RA, and positive selection or enrichment for cells expressing CD62L. In one aspect, enrichment

Central memory T cells (TCM) are performed starting with a negative fraction of cells selected based on the expression of CD4, which is subjected to a negative selection based on the expression of CD14 and CD45RA, and the positive selection with based on CD62L. Such selections, in some aspects, are made simultaneously and in other aspects, they are made sequentially, in any order. In some respects, the same selection step based on the CD4 expression used in the preparation of the CD8 + cell subpopulation or population is also used to generate the CD4 + cell subpopulation or population, such that both positive and negative fractions of the separation based on CD4 are retained and used in the subsequent steps of the methods, optionally after one or more other steps of positive or negative selection.

[00598] In a particular example, a sample of PBMCs or another sample of white blood cell is subjected to selection of CD4 + cells, where both negative and positive fractions are retained. The negative fraction is then subjected to negative selection based on the expression of CD14 and CD45RA or ROR1, and positive selection based on a marker characteristic of central memory T cells, such as CD62L or CCR7, where the selections positive and negative are carried out in any order.

[00599] CD4 + T helper cells are classified into virgin, central memory and effector cells by identifying cell populations that have cell surface antigens. CD4 + lymphocytes can be obtained by standard methods. In some modalities, virgin CD4 + T lymphocytes are CD45RO-, CD45RA +, CD62L +, CD4 + T cells. In some embodiments, the central memory CD4 + cells are CD62L + and CD45RO +. In some modalities, CD4 + effector cells are CD62L- and CD45RO-.

[00600] In one example, enriching for CD4 + cells by negative selection, a monoclonal antibody cocktail typically includes antibodies to CD14, CD20, CD11b, CD16, HLA-DR, and CD8. In some embodiments, the antibody or binding partner is attached to a solid support or matrix, such as a magnetic bead or paramagnetic bead, to allow separation of cells by positive and / or negative selection. For example, in some embodiments, cells and cell populations are separated or isolated using immunomagnetic separation techniques (or magnetic affinity) (reviewed in Methods in Molecular Medicine, vol. 58: Metastasis Research Tools, Vol 2: Cell Behavior In vitro and In vivo, p 17-25 Edited by: SA Brooks and U. Schumacher © Humana Press Inc., Totowa, NJ).

[00601] In some respects, the sample or cell composition to be separated is incubated with small, magnetizable or magnetically responsive material, such as microparticles or magnetically responsive particles, such as paramagnetic beads (for example, such as Dynalbeads beads or MACS). The magnetically responsive material, for example, particle, is usually directly or indirectly linked to a binding partner, for example, an antibody, which specifically binds to a molecule, for example, surface marker, present in cells, cells, or cell population that is desired to separate, for example, that is desired to select negatively or positively.

[00602] In some embodiments, the magnetic bead or particular comprises a magnetically responsive material attached to a specific binding member, such as an antibody or other binding partner. There are many well known magnetically responsive materials used in magnetic separation methods. Suitable magnetic particles include those described in Moled, United States Patent No. 4,452,773, and in European Patent Specification EP 452342 B, which are incorporated into this document.

document by reference. Colloidal size particles, such as those described in U.S. Patent No. 4,795,698, and Liberti and others, U.S. Patent No. 5,200,084 are other examples.

[00603] Incubation is generally performed under conditions in which antibodies or binding partners, or molecules, such as secondary antibodies or other agents, that specifically bind to such antibodies or binding partners, which are bound to the magnetic bead or particle, they specifically bind to the cell surface molecules, if present in the cells within the sample.

[00604] In some aspects, the sample is placed in a magnetic field, and those cells having magnetically responsive or magnetizable particles attached to it will be attached to the magnet and separated from the unlabeled cells. For positive selection, the cells that are attached to the magnet are retained; for negative selection, cells that are not bound (unlabeled cells) are retained. In some aspects, a combination of positive and negative selection is carried out during the same selection step, where positive and negative fractions are retained and also processed or subjected to other separation steps.

[00605] In certain embodiments, the magnetically responsive particles are coated with primary antibodies or other binding partners, secondary antibodies, lectins, enzymes, or streptavidin. In certain embodiments, the magnetic particles are attached to the cells by means of a coating of primary antibodies specific for one or more markers. In certain embodiments, cells, unlike beads, are labeled with a primary antibody or binding partner, and then cell type-specific secondary antibody or other magnetic particles coated with binding partner (eg streptavidin) , are added. In certain modalities

Streptavidin-coated magnetic particles are used in conjunction with biotinylated primary or secondary antibodies.

[00606] In some embodiments, the magnetically responsive particles are left attached to the cells that must be subsequently incubated, cultured and / or modified; in some aspects, the particles are left attached to the cells for administration to the patient. In some embodiments, the magnetically responsive or magnetizable particles are removed from the cells. Methods for removing magnetizable particles from cells are known and include, for example, the use of non-labeled competition antibodies, magnetizable particles or antibodies conjugated to cleavable linkers, etc. In some embodiments, the magnetizable particles are biodegradable.

[00607] In some modalities, selection based on affinity is by means of magnetic activated cell classification (MACS) (Miletten Biotec, Auburn, CA). Magnetic Activated Cell Classification (MACS) systems are capable of high-purity selection of cells having magnetizable particles attached to them. In certain modes, MACS operates in a mode in which non-target and target species are sequentially eluted after the application of the external magnetic field. That is, cells attached to the magnetized particles are held in place while unbound species are eluted. Then, after this first elution step is completed, the species that have been trapped in the magnetic field and prevented from being eluted are released in some way that they can be eluted and recovered. In certain respects, non-target cells are labeled and depleted from the heterogeneous population of cells.

[00608] In certain modalities, isolation or separation is performed using a system, device or apparatus that performs one or more of the isolation, cell preparation, separation, processing, incubation, culture and / or formulation steps methods. In some respects, the system is used to perform each of these steps in a closed or sterile environment, for example, to minimize errors, user manipulation and / or contamination. In one example, the system is a system as described in international PCT Publication No. WO 2009/072003, or US 20110003380 A1.

[00609] In some modalities, the system or apparatus performs one or more, for example, all, of the isolation, processing, modification and formulation steps in an integrated or independent system and / or in an automated or programmable pattern . In some aspects, the system or apparatus includes a computer and / or computer program in communication with the system or apparatus, which allows a user to program, control, evaluate the result of, and / or adjust various aspects of processing steps, isolation, modification and formulation.

[00610] In some aspects, separation and / or other steps are performed using the CliniMACS system (Miltenyi Biotec), for example, for automated separation of cells at a clinical scale level in a closed system and sterile. Components can include an integrated microcomputer, magnetic separation unit, peristaltic pump and several pinch valves. The integrated computer, in some aspects, controls all components of the instrument and directs the system to perform repeated procedures in a standardized sequence. The magnetic separation unit, in some aspects, includes a movable permanent magnet and a support for the selection column. The peristaltic pump controls the flow rate throughout the set of tubes and, together with the clamping valves, ensures the controlled flow of buffer through the system and continuous cell suspension.

[00611] The CliniMACS system, in some aspects, uses magnetizable particles coupled to the antibody that are supplied in a sterile non-pyrogenic solution. In some embodiments, after labeling the cells with magnetic particles, the cells are washed to remove excess particles. A cell preparation bag is then connected to the tube group, which in turn is connected to a bag containing a buffer and a cell collection bag. The tube group consists of pre-assembled sterile tubes, including a precolumn and a separation column and are for single use only. After starting the separation program, the system automatically applies the cell sample to the separation column. The labeled cells are retained within the column, while the unlabeled cells are removed by a series of washing steps. In some embodiments, cell populations for use with the methods described in this document are unlabeled and are not retained in the column. In some modalities, cell populations for use with the methods described in this document are labeled and are retained in the column. In some modalities, cell populations for use with the methods described in this document are eluted from the column after removal of the magnetic field, and are collected inside the cell collection bag.

[00612] In certain modalities, the separation and / or other steps are performed using the CliniMACS Prodigy system (Miltenyi Bio-tec). The CliniMACS Prodigy system, in some aspects, is equipped with a cell processing unit that allows automatic washing and cell fractionation by centrifugation. The CliniMACS Prodigy system can also include an onboard camera and image recognition software that determines the end point of optimal cell fractionation by discerning the macroscopic layers of the source cell product. For example, peripheral blood can be

automatically separated into erythrocytes, white blood cells and layers of plasma. The CliniMACS Prodigy system can also include an integrated cell culture chamber that performs cell culture protocols such as, for example, cell expansion and differentiation, antigen loading, and long-term cell culture. The entrance doors can allow sterile removal and the replacement of media and cells can be monitored using an integrated microscope. See, for example, Klebanoff and others (2012) J Immunother. 35 (9): 651–660, Terakura and other (2012) Blood.1: 72–82, and Wang and other (2012) J Immunother.35 (9): 689-701.

[00613] In some embodiments, a cell population described in this document is collected and enriched (or depleted) by means of flow cytometry, in which cells stained by multiple cell surface markers are loaded in a fluidic stream . In some modalities, a cell population described in this document is collected and enriched (or depleted) by means of a preparatory scale classification (FACS). In certain modes, a cell population described in this document is collected and enriched (or depleted) by the use of microcelectromechanical system (MEMS) chips in combination with a FACS-based detection system (see , for example, WO 2010/033140, Cho et al. (2010) Lab Chip 10: 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5): 355–376). In both cases, cells can be labeled with multiple markers, allowing the isolation of well-defined T cell subgroups in high purity.

[00614] In some embodiments, antibodies or binding partners are labeled with one or more detectable markers, to facilitate separation by positive and / or negative selection. For example, separation can be based on binding to fluorescently labeled antibodies. In some examples, the separation of cells based on the binding of antibodies or other binding partners specific to one or more cell surface markers is loaded into a fluidic stream, such as by fluorescence-activated cell classification (FACS), including preparative scale (FACS) and / or microelectromechanical system chips (MEMS), for example, in combination with a flow cytometry detection system. Such methods allow for positive and negative selection based on multiple markers simultaneously.

[00615] In some modalities, the preparation methods include the steps for freezing, for example, cryopreservation, of the cells, before or after isolation, incubation and / or modification. In some embodiments, freezing and the subsequent thawing step remove granulocytes and, to some extent, monocytes in the cell population. In some embodiments, the cells are suspended in a freezing solution, for example, after a washing step to remove plasma and platelets. Any of a variety of known freezing solutions and parameters, in some ways, can be used. One example involves using PBS that contains 20% DMSO and 8% human serum albumin (HSA), or other suitable cell freezing means. This is then diluted 1: 1 with means so that the final concentration of DMSO and HSA is 10% and 4%, respectively. The cells are then frozen at −80 ° C at a rate of 1 ° per minute and stored in the vapor phase of a liquid nitrogen storage tank.

[00616] In some modalities, the methods provided include stages of cultivation, incubation, culture, and / or genetic modification. For example, in some modalities, provided are methods for incubating and / or modifying depleted cell populations and culture initiation compositions.

[00617] Thus, in some modalities, cell populations are incubated in a culture initiation composition. Incubation and / or modification can be performed in a culture vessel, such as a unit, chamber, cavity, column, tube, group of tubes, valve, flask, culture disc, bag, or other container for cells of culture or cultivation.

[00618] In some embodiments, cells are incubated and / or cultured before or in connection with genetic modification. Incubation steps can include culture, cultivation, stimulation, activation, and / or propagation. In some embodiments, the compositions or cells are incubated in the presence of stimulating conditions or a stimulating agent. Such conditions include those designed to induce cell proliferation, expansion, activation, and / or survival in the population, for mimic antigen exposure, and / or to prepare cells for genetic modification, such as for the introduction of nucleic acids that encodes a recombinant receptor, for example, a recombinant TCR.

[00619] Conditions may include one or more of the particular media, temperature, oxygen content, carbon dioxide content, time, agents, for example, nutrients, amino acids, antibiotics, ions and / or stimulatory factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, matching soluble receptors, and any other agents designed to activate cells.

[00620] In some embodiments, the stimulating conditions or agents include one or more agents, for example, ligand, which is capable of activating an intracellular signaling region of a TCR complex. In some respects, the agent binds or initiates the TCR / CD3 intracellular signaling cascade in a T cell. Such agents may include antibodies, such as those specific for a TCR, for example, anti-CD3. In some embodiments, the stimulating conditions include one or more agents, for example, ligand, which is capable of stimulating a co-stimulatory receptor, for example, anti-CD28. In some embodiments, such agents and / or binders can be attached to the solid support such as a bead and / or one or more cytokines. Optionally, the expansion method may also comprise the step of adding anti-CD3 and / or anti CD28 antibody to the culture medium (for example, at a concentration of at least about 0.5 ng / ml). In some embodiments, stimulating agents include IL-2, IL-15 and / or IL-7. In some respects, the IL-2 concentration is at least about 10 units / ml.

[00621] In some respects, incubation is performed according to techniques such as those described in US Patent No. 6,040,177 to Riddell and others (Klebanoff and others (2012) J Immunother. 35 (9): 651–660, Terakura and others (2012) Blod od.1: 72–82, and / or Wang and others (2012) J Immunother. 35 (9): 689-

701.

[00622] In some embodiments, T cells are expanded by adding to the culture initiation composition feeder cells, such as non-divisible peripheral blood mononuclear cells (PBMC), (for example, such that the resulting population of cells contain at least about 5, 10, 20, or 40 or more PBMC feeder cells for each T lymphocyte in the initial population to be expanded); and incubating the culture (for example, long enough to expand T cell numbers). In some respects, non-divisible feeder cells may comprise gamma-irradiated PBMC feeder cells. In some modes, PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some respects, feeder cells are added to the culture medium prior to the addition of T cell populations.

[00623] In some embodiments, the stimulating conditions included at an appropriate temperature for the growth of human T lymphocytes, for example, at least about 25 degrees Celsius, generally at least about 30 degrees, and generally at or about 37 degrees Celsius. Optionally, the incubation can also comprise adding non-divisible EBV-transformed lymphoblastoid cells (LCL) as feeder cells. LCL can be irradiated with gamma rays in the range of about 6000 to 10,000 rads. LCL feeder cells, in some respects, are provided in any suitable amount, such as a ratio of LCL feeder cells to the initial T lymphocytes of at least about 10: 1.

[00624] In some embodiments, antigen-specific T cells, such as CD4 + and / or CD8 + antigen-specific T cells, are obtained by stimulating antigen-specific or virgin T-lymphocytes with antigen. For example, antigen-specific T cell clones or strains can be generated for cytomegalovirus antigens by isolating T cells from infected individuals and stimulating cells in vitro with the same antigen.

[00625] Various methods for introducing genetically modified components, for example, agents to induce a genetic disruption and / or nucleic acids encoding recombinant receptors, for example, CARs or TCRs, are known and can be used with the methods and compositions provided. Exemplary methods include those for transferring nucleic acids encoding polypeptides or receptors, including via viral vectors, for example, non-viral, retroviral or lentiviral vectors or transposons, for example, Transposon Sleeping Beauty System. Gene transfer methods may include transduction, electroporation, or another method that results in gene transfer in the cell, or any release methods described in Section I.A. Other methods and vectors for transferring nucleic acids encoding recombinant products are those described, for example, in WO 2014055668 and in U.S. Patent No. 7,446,190.

[00626] In some embodiments, recombinant nucleic acids are transferred to T cells via electroporation (see, for example, Chicaybam et al, (2013) PLoS ONE 8 (3): e60298 and Van Teloelo et al. (2000 ) Gene Therapy 7 (16): 1431-1437). In some modalities, recombinant nucleic acids are transferred to T cells through transposition (see, for example, Manuri et al. (2010) Hum Gene Ther 21 (4): 427-437; Sharma et al. ( 2013) Molec Ther Nucl Acids 2, e74; and Huang et al. (2009) Methods Mol Biol 506: 115-126). Other methods of introducing and expressing genetic material into immune cells include calcium phosphate transfection (for example, as described in Current Protocols in Molecular Biology, John Wiley & Sons, New York. NY), protoplast fusion, cationic liposome-mediated transfection ; bombardment of microparticles facilitated by tungsten particles (Johnston, Nature, 346: 776-777 (1990)); and strontium phosphate DNA co-precipitation (Brash et al., Mol. Cell Biol., 7: 2031-2034 (1987)).

[00627] In some embodiments, gene transfer is performed by first stimulating the cell, such as combining it with a stimulus that induces a response such as proliferation, survival, and / or activation, for example, measured by the expression of a cytokine or activation marker, followed by transduction of activated cells, and expansion in culture to sufficient numbers for clinical applications.

[00628] In some contexts, it may be desirable to safeguard against the potential of this overexpression of a stimulating factor (for example, a lymphokine or a cytokine) that could potentially result in an undesired result or less effectiveness in an individual, such as a factor associated with toxicity in an individual. Thus, in some contexts, the modified cells include segments of genes that make the cells susceptible to negative selection in vivo, such as after administration in adoptive immunotherapy. For example, in some ways, cells are modified so that they can be eliminated as a result of a change in the in vivo condition of the patient to whom they are administered. The selectable negative phenotype can result from the insertion of a gene that confers sensitivity to an administered agent, for example, a compound. Selectable negative genes include the Herpes simplex virus type I thymidine kinase (HSV-I TK) gene (Wigler et al., Cell 11: 223, 1977) which confers sensitivity to ganciclovir; the cell hypoxanthine phosphoribosyltransferase (HPRT) gene, the cell adenine phosphoribosyl transferase (APRT) gene, bacterial cytosine deaminase (Mullen et al., Proc. Natl. Acad. Sci. USA. 89:33 (1992)).

[00629] In some embodiments, cells, for example, T cells, can be modified during or after expansion. Such modification for an introduction of the desired polypeptide or receptor gene can be carried out with any suitable retroviral vector, for example. The population of genetically modified cells can then be released from the initial stimulus (the CD3 / CD28 stimulus, for example) and subsequently be stimulated with a second type of stimulus (for example, via a receptor introduced from New). This second type of stimulus can include an antigenic stimulus in the form of a peptide / MHC molecule, the cognate ligand (crosslinking) of the genetically introduced receptor (for example, natural ligand from a CAR) or any ligand (such as an antibody) that binds directly within the structure of the new receptor (for example, recognizing constant regions within the receptor). See, for example,

Cheadle et al, "Chimeric antigen receptors for T-cell based therapy" Methods Mol Biol. 2012; 907: 645-66 or Barrett et al., Chimeric Antigen Receptor Therapy for Cancer Annual Review of Medicine Vol. 65: 333- 347 (2014).

[00630] Among additional nucleic acids, for example, the genes for introduction are those that improve the effectiveness of therapy, such as promoting the viability and / or function of the transferred cells; genes to provide a genetic marker for selection and / or evaluation of cells, such as evaluating survival or in vivo location; genes to improve safety, for example, making the cell susceptible to negative selection in vivo, as described by Lupton S. D. et al., Mol. and Cell Biol., 11: 6 (1991); and Riddell et al., Human Gene Therapy 3: 319-338 (1992); see also PCT / US91 / 08442 and PCT / US94 / 05601 publications by Lupton et al. describing the use of selectable bifunctional fusion genes derived from the fusion of a dominant positive selectable marker with a negative selectable marker. See, for example, Riddell et al., U.S. Patent No. 6,040,177, in columns 14 to 17.

[00631] As described in this document, in some ways, cells are incubated and / or cultured before or in connection with genetic engineering. Incubation steps can include culture, cultivation, stimulation, activation, propagation and / or freezing for preservation, for example, cryopreservation. IV. RECOMBINANT T-CELL RECEPTORS (TCRs)

[00632] In some embodiments, the recombinant receptor that is introduced into the cell is a T cell receptor (TCR) or an antigen binding fragment thereof.

[00633] In some embodiments, the transgene for targeted integration encodes a recombinant receptor or an antigen-binding fragment of the same or a chain thereof. In some embodiments, the recombinant receptor is a recombinant antigen receptor, or a recombinant receptor that binds to an antigen. In some embodiments, the recombinant receptor is a recombinant or modified T cell (TCR) receptor. In some respects, the recombinant TCR is different from the endogenous TCR encoded by the T cell. In some embodiments, the polynucleotides, vectors, compositions, methods, manufactured articles provided, and / or kits are useful for modifying cells that express a recombinant TCR or antigen-binding fragment thereof.

[00634] In some embodiments, the provided recombinant receptors, for example, TCRs or CARs, are capable of binding to or recognizing, such as specifically binding to or recognizing, an antigen that is associated with, specific for, and / or expressed in a cell or tissue of a disease, disorder or condition, such as a cancer or tumor. In some ways, the antigen is in the form of a peptide, for example, it is a peptide antigen or a peptide epitope. In some embodiments, the TCRs provided bind to, as they specifically bind to, an antigen that is a peptide, in the context of a molecule of greater histocompatibility (MHC).

[00635] The observation that the recombinant receptor binds to an antigen, for example, peptide antigen, or specifically binds to an antigen, for example, peptide antigen, does not necessarily mean that it binds to an antigen of all species. For example, in some embodiments, antigen-binding characteristics, for example, peptide antigen in the context of an MHC, such as the ability to specifically bind to it and / or compete for binding with a reference binding molecule or a receptor, and / or to bind to a particular affinity or compete to a certain degree, in some modalities, refers to the ability with respect to a human antigen and the recombinant receptor that may not have this characteristic in relation to the antigen of another species, such as a mouse. In some respects, the extent of binding of the recombinant receptor or an antigen-binding fragment thereof to an unrelated antigen or protein, such as an unrelated peptide antigen, is less than or about 10% of the binding recombinant receptor or antigen-binding fragment thereof to the antigen, for example, cognate antigen as measured, for example, by a radioimmunoassay (RIA), a peptide titration assay or a reporter assay. A. T cell receptors (TCRs)

[00636] In some embodiments, a "T cell receptor" or "TCR" is a molecule that contains α and β chains (also known as TCRα and TCRβ, respectively) or variable γ and δ chains (also known as TCRγ and TCRδ , respectively), or antigen-binding parts thereof, and which is capable of specifically binding to an antigen, for example, a peptide antigen or peptide epitope, attached to an MHC molecule. In some modalities, the TCR is in αβ form. Typically, the TCRs that exist in αβ and γδ forms are generally structurally similar, but the T cells that express them may have different anatomical functions or locations. A TCR can be found on the surface of a cell or in soluble form. Generally, a TCR is found on the surface of T cells (or T lymphocytes) where it is generally responsible for the recognition of antigens linked to the molecules of the major histocompatibility complex (MHC). In some embodiments, the antigen that is bound or recognized, as specifically bound or recognized, by the TCR is a peptide. In some respects, the peptide that is linked or recognized, as specifically linked or recognized, by the TCR is an antigen peptide. In some ways, "antigen" attached to or recognized by a TCR, includes a peptide.

[00637] Normally, specific binding of the recombinant receptor, for example, TCR, to a peptide epitope, for example, in complex with an MHC, is governed by the presence of an antigen-binding site that contains one or more complementarity determining regions (CDRs). In general, it is understood that binding specifically does not mean that the particular peptide epitope, for example, in complex with an MHC, is the only thing that the MHC peptide molecule can bind to, since that nonspecific binding interactions with other molecules can also occur. In some embodiments, binding of the recombinant receptor to a peptide in the context of an MHC molecule is of greater affinity than binding to such other molecules, for example, another peptide in the context of an MHC molecule or a peptide irrelevant (control) in the context of an MHC Molecule, such as at least about 2 times, at least about 10 times, at least about 20 times, at least about 50 times, or at least about 100 times greater than the binding affinity for these other molecules.

[00638] In some embodiments, recombinant receptor, for example, TCR, can be assessed for safety or off-target binding activity using any of a series of known screening assays. In some embodiments, the generation of an immune response to a particular recombinant receptor, for example, TCR, can be measured in the presence of cells that do not express the epitope of the target peptide, such as cells derived from normal tissue (s) ( is), allogeneic cell lines that express one or more different types of MHC or other tissues or cell sources. In some embodiments, the cells or tissues include normal cells or tissues. In some embodiments, cell binding can be tested in two-dimensional cultures. In some embodiments, cell binding can be tested in three-dimensional cultures.

In some embodiments, such as a control, the tissues or cells may be those that are known to express the target epitope. The immune response can be assessed directly or indirectly, such as assessing activation of immune cells such as T cells (eg, cytotoxic activity), cytokine production (eg, gamma interferon), or activation of a signaling cascade , as well as reporter tests.

[00639] Unless otherwise indicated, the term "TCR" should be understood to encompass complete TCRs, as well as antigen-binding portions or antigen-binding fragment thereof. In some embodiments, the TCR is an intact or complete TCR, such as a TCR that contains the alpha (α) chain and the beta (β) chain. In some embodiments, the TCR is an antigen-binding portion that is less than a full-length TCR, but that binds to a specific peptide attached to an MHC molecule, just as it binds to a peptide complex. of MHC. In some cases, an antigen-binding portion or TCR fragment may define only a portion of the structural domains of an entire or whole TCR, but is still capable of binding to the peptide epitope, such as the peptide complex of MHC, to which the complete TCR binds. In some cases, an antigen-binding portion contains the variable domains of a TCR, such as the variable α (Vα) chain and the variable β (V β) chain of a TCR, or antigen-binding fragment thereof, sufficient to form a binding site for binding to a specific MHC peptide complex.

[00640] In some embodiments, the variable domains of the TCR contain complementarity determining regions (CDRs), which are generally the primary contributors to antigen recognition and binding capabilities and specificity of the MHC peptide, MHC and / or peptide complex . In some modalities, a

CDRs from a TCR or combination thereof form all or substantially all the antigen-binding sites of a given TCR molecule. The various CDRs within a variable region of a TCR chain are usually separated by structural regions (FRs), which generally exhibit less variability between TCR molecules compared to CDRs (see, for example, Jores et al., Proc. Nat'l Acad. Sci. USA 87: 9138, 1990; Chothia et al., EM-BO J. 7: 3745, 1988; see also Lefranc et al., Dev. Comp. Immunol. 27: 55, 2003). In some modalities, CDR3 is the main CDR responsible for the binding or specificity to the antigen, or is the most important among the three CDRs in a given variable region of the TCR for recognition of the antigen, and / or for interaction with the portion processed peptide from the MHC peptide complex. In some contexts, CDR1 of the α chain can interact with the N-terminal part of certain antigenic peptides. In some contexts, CDR1 of the β chain can interact with the C-terminal part of the peptide. In some contexts, CDR2 contributes more strongly to or is the primary CDR responsible for interacting with or recognizing the MHC portion of the MHC peptide complex. In some modalities, the variable region of the β chain can determine an additional hypervariable region (CDR4 or HVR4), which is generally involved in the binding of the superantigen and non-recognition of the antigen (Kotb (1995) Clinical Microbiology Reviews, 8: 411- 426).

[00641] In some embodiments, the TCRα chain and / or TCR chain of a TCR also contains a constant domain, a transmembrane domain and / or a short cytoplasmic tail (see, for example, Janeway et al., Immunobiology: The Immune System in Health and Disease, 3rd edition., Current Biology Publications, p. 4:33, 1997). In some respects, each TCR chain (for example, alpha or beta) may have an N-terminal immunoglobulin variable domain, a

constant immunoglobulin domain, a transmembrane region and a short cytoplasmic tail at the C-terminal end. In some modalities, a TCR, for example, by means of the cytoplasmic tail, is associated with invariant proteins of the CD3 complex involved in the mediation of signal transduction. In some cases, the structure allows the TCR to associate with other molecules such as CD3 and its subunits. For example, a TCR that contains constant domains with a transmembrane region can anchor protein in the cell membrane and associate with invariant subunits of the CD3 complex or signaling device. The intracellular tails of the CD3 signaling subunits (for example, CD3γ, CD3δ, CD3ε and CD3ζ chains) contain one or more activation motifs based on immunoreceptor tyrosine or ITAM and are generally involved in the signaling ability of the TCR complex.

[00642] In some embodiments, the various domains or regions of a TCR can be determined. In some cases, the exact locus of a domain or region can vary depending on the structural modeling or particular homology or other characteristics used to describe a particular domain. It is understood that reference to amino acids, including a specific sequence presented as a SEQ ID NO used to describe the domain organization of a recombinant receptor, for example, TCR, is for illustrative purposes and is not intended to limit the scope of the modalities provided. In some cases, the specific domain (for example, variable or constant) can be several amino acids (such as one, two, three or four) longer or shorter. In some respects, residues from a TCR are known or can be identified according to the numbering system of the International Immunogenetic Information System (IMGT) (see, for example, www.imgt.org; see also, Lefranc et al. (2003) Developmental and Comparative Immunology, 2 &55-77; and

The T Cell Factsbook 2nd Edition, Lefranc and LeFranc Academic Press 2001). Using this system, the CDR1 sequences within a TCR Vα chain and / or Vβ chain correspond to the amino acids present between residues numbers 27 to 38, including the CDR2 sequences within a TCR Vα chain and / or Vβ chain correspond to the amino acids present between residues numbers 56 to 65, inclusive, and the CDR3 sequences within a TCR Vα chain and / or Vβ chain correspond to the amino acids present between residues numbers 105 to 117, inclusive.

[00643] In some modalities, the α chain and the β chain of a TCR, each one, still, assigns a constant domain. In some embodiments, the constant domain of the α (Cα) chain and the constant domain of the β (Cβ) chain are mammals, just as it is a human or murine constant domain. In some embodiments, the constant domain is adjacent to the cell membrane. For example, in some cases, the extracellular portion of the TCR formed by the two chains contains two constant domains proximal to the membrane, and two variable domains distal to the membrane, where each variable domain contains CDRs.

[00644] In some modalities, each of the constant domains TCRα and TCRβ is human. In some modalities, Cα is encoded by the TRAC gene (IMGT nomenclature) or is a variant thereof. In some embodiments, Cα has or comprises the amino acid sequence set out in SEQ ID NO: 19 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 19. In some modalities, Cα has or comprises the amino acid sequence established in any of SEQ ID NO: 19. In some embodiments, Cα has or comprises the amino acid sequence, for example,

mature polypeptide, encoded by a nucleic acid sequence established in SEQ ID NO: 1 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the amino acid sequence, for example, mature polypeptide, encoded by a nucleic acid sequence set out in SEQ ID NO :1. In some modalities, C is encoded by TRBC1 or TRBC2 genes (IMGT nomenclature) or is a variant thereof. In some embodiments, C has or comprises the amino acid sequence set out in SEQ ID NO: 20 or 21 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90 %, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 20 or 21. In some embodiments, C has or comprises the amino acid sequence set out in SEQ ID NO: 20 or 21. In some embodiments, C has or comprises the amino acid sequence, for example, mature polypeptide, encoded by a nucleic acid sequence set out in SEQ ID NO : 2 or 3 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more sequence identity to the amino acid sequence, for example, mature polypeptide, encoded by a nucleic acid sequence established in SEQ ID NO: 2 or 3.

[00645] In some embodiments, a portion of the recombinant TCR is encoded by at least part of the open reading frame of a TRAC or TRBC gene. In certain modalities, releasing, introducing, contacting, transfecting, and / or transducing the transgene, polynucleotide or vector to a cell results in expression of the recombinant TCR. In some embodiments, a portion of the recombinant TCR is encoded by the transgene, polynucleotide, and / or vector, and the remaining portion is encoded by a nucleic acid sequence that is endogenous and / or native to the cell. In certain embodiments, the endogenous and / or native nucleic acid sequence is or is within an open reading frame of a TRAC or TRBC locus. In some embodiments, the TRAC or TRBC locus is human. In particular modalities, the remaining portion of the TCR is a constant domain, for example, a constant domain TCRα. or TCRβ. In certain modalities, the remaining portion of the TCR is a part of a constant domain, for example, a part of the constant domain TCRα. or TCRβ.

[00646] In some embodiments, any of the supplied TCRs or antigen-binding fragment thereof may be a human / mouse chimeric TCR. In some cases, the TCR or antigen-binding fragment of the same has an α chain and / or a β chain that comprises a constant mouse region. In some respects, the Cα and / or Cβ regions are constant mouse regions. In some modalities, Cα is a mouse constant region that is or comprises the amino acid sequence set out in SEQ ID NO: 14, 15, 121 or 122 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 14 , 15, 121 or 122. In some modalities, Cα is or comprises the amino acid sequence set out in SEQ ID NO: 14, 15, 121 or 122. In some modalities, C is a constant region of mouse that is or comprises the amino acid sequence set out in SEQ ID NO: 16, 17 or 123 or an amino acid sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90% , 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 16, 17 or 123. In some embodiments, C  is or comprises the amino acid sequence set out in SEQ ID NO: 16, 17 or

123.

[00647] In some of these modalities, the TCR or antigen-binding fragment thereof contains one or more modifications in the α and / or β-chain such that when the TCR or antigen-binding fragment is expressed in a cell, the frequency of incorrect pairing between the α and β chain of the TCR and an α and β chain of the endogenous TCR is reduced, the expression of the α chain and the β chain of the TCR is increased and / or the stability of the α chain and the β chain of the TCR is increased. In some modalities, one or more modifications is a substitution, deletion, or insertion of one or more amino acids in the Cα region and / or in the Cβ region. In some respects, the one or more modifications contain substitution (s) to introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the α chain and the β chain.

[00648] In some of such modalities, the TCR or antigen-binding fragment thereof that contains a Cα region that contains a cysteine in a position that corresponds to position 48 with numbering as mentioned in SEQ ID NO: 24 e / or a Cβ region that contains a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO: 20. In some embodiments, said Cα region contains the amino acid sequence established in any of the SEQs ID NOS: 19 or 24, or an amino acid sequence that has at least 90% sequence identity with the same that contains one or more cysteine residues capable of forming a non-native disulfide bond with the β chain; and / or said Cβ region contains the amino acid sequence established in any of SEQ ID NOS: 20, 21 or 25, or an amino acid sequence that has at least 90% sequence identity that contains one or more residues of cysteine capable of forming a non-native disulfide bond with the α chain.

[00649] In some of such modalities, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that has been codon-optimized.

[00650] In some of such modalities, the binding molecule or TCR or antigen-binding fragment thereof is isolated or purified or is recombinant. In some of these modalities, the binding molecule or TCR or antigen binding fragment thereof is human.

[00651] In some embodiments, the TCR can be a heterodimer of two α and β chains that are linked, such as by a disulfide bond or disulfide bonds. In some embodiments, the TCR constant domain can alter short connection sequences in which a cysteine residue forms a disulfide bond, thereby connecting the two TCR chains. In some embodiments, a TCR may have an additional cysteine residue in each of the α and β chains, so that the TCR contains two disulfide bonds in the constant domains. In some embodiments, each of the constant and variable domains contains disulfide bonds formed by cysteine residues.

[00652] In some embodiments, the TCR may contain an introduced disulfide bond or bonds. In some embodiments, native disulfide bonds are not present. In some modalities, one or more of the native cysteines (for example, in the constant domain of the α chain and the β chain) that form a native interchain disulfide bond are replaced by another residue, such as a serine or alanine . In some embodiments, an introduced disulfide bond may be formed by mutating non-cysteine residues in the alpha and β chains, such as in the constant domain of the α and β chain, in cysteine. In some embodiments, the presence of non-native cysteine residues (for example, resulting in one or more non-native disulfide bonds) in a recombinant TCR can favor the production of the desired recombinant TCR in a cell in which it is introduced over the expression of an incomparable TCR pair that contains a native TCR chain.

[00653] Non-native disulfide bonds exemplary of a TCR are described in PCT International No. WO 2006/000830, WO 2006037960 and Kuball et al. (2007) Blood, 109: 2331-2338. In some modalities, cysteines can be introduced in the Thr48 residue of the Cα chain and Ser57 of the Cβ chain, in the Thr45 residue of the Cα chain and Ser77 of the Cβ chain, in the Tyr10 residue of the Cα chain and Ser17 of the Cβ chain, in the Thr45 residue of the Cα and Asp59 chain of the Cβ chain and / or in the Ser15 residue of the Cα chain and Glu15 of the Cβ chain with reference to the numbering of a Cα established in SEQ ID NO: 24 or Cβ established in SEQ ID NO: 20. In some modalities, any of the cysteine mutations provided can be done in a corresponding position in another sequence, for example, in the mouse Cα and Cβ sequences described in this document. The term "corresponding" with reference to the positions of a protein, such as reciting that the positions of amino acids "correspond to" positions of amino acids in a described sequence, as established in a sequence listing, refers to the positions of amino acids identified after alignment with the described sequence based on structural sequence alignment or using a standard alignment algorithm, such as the GAP algorithm. For example, the corresponding residues can be determined by aligning a reference sequence with the Cα sequence established in any one of SEQ ID NO: 24 or the Cβ sequence established in SEQ ID NO: 20 by structural alignment methods as described - described in this document. When aligning the sequences, the corresponding residues can be identified, for example, using

pairs of conserved and identical amino acids as guides.

[00654] Exemplary sequences (for example, CDRs, Vα and / or V and regions of constant sequences) of the TCRs provided are described in this document.

[00655] In some embodiments, the recombinant TCR or antigen-binding portion of the same (or another MHC peptide-binding molecule, such as TCR-type antibody) is known or can recognize a peptide epitope or T cell epitope from a target polypeptide when presented by cells in the context of an MHC molecule, i.e., MHC peptide complex of the target polypeptide. In some embodiments, the recombinant TCR (or other MHC peptide-binding molecule or TCR-like antibody) is known to exhibit specific binding to the T cell epitope of the target polypeptide, for example, when displayed as a peptide complex of MHC. Methods for assessing the binding or interaction of an MHC peptide binding molecule (for example, TCR or TCR-like antibody) are known, including any of the exemplary methods described herein.

[00656] In some embodiments, the MHC molecule is either a class I MHC molecule or a class II MHC molecule. In some embodiments, the MHC contains a polymorphic peptide binding site or binding groove that can, in some cases, complement polypeptide peptide epitopes, including peptide epitopes processed by cellular machinery. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a peptide complex, that is, MHC peptide complex, for presenting an antigen in a TCR-recognizable configuration in T cells, or other MHC peptide binding molecules. Generally, MHC class I molecules are heterodimers with a membrane spanning the α chain, in some cases with three α domains, and a non-covalently associated β2 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which normally cross the membrane. An MHC molecule can include an effective portion of an MHC that contains an antigen binding site or sites for the binding of a peptide and the sequences necessary for recognition by the appropriate binding molecule, such as the TCR. In some embodiments, MHC class I molecules release peptides originating from cytosol to the cell surface, where a peptide: MHC complex is recognized by T cells, as usually CD8 + T cells, but in some cases CD4 + T cells . In some embodiments, MHC class II molecules release peptides from the vesicular system to the cell surface, where they are typically recognized by CD4 + T cells. Generally, MHC molecules are encoded by a group of linked loci, which are collectively called H-2 in mice and human leukocyte antigen (HLA) in humans. In some ways, human MHC can also be referred to as a human leukocyte antigen (HLA).

[00657] In some embodiments, the peptide epitope or T cell epitope is a peptide that can be derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein, and which is capable of to associate with or form a complex with an MHC molecule. In some embodiments, the peptide is about 8 to about 24 amino acids in length. In some embodiments, the peptide is about 9 to 22 amino acids long for recognition in the Class II MHC complex. In some embodiments, the peptide is about 8 to 13 amino acids long for recognition in the MHC Class I complex. In some embodiments, the MHC molecule and the peptide epitope or T cell epitope are complexed or associated with means of non-covalent interactions of the peptide in the MHC molecule binding groove or gap.

[00658] In some embodiments, the MHC peptide complex is present or displayed on the surface of cells. In some embodiments, the MHC peptide complex can be recognized specifically by a TCR or antigen binding moiety in the same way, or another MHC peptide binding molecule. In some embodiments, the T cell epitope or peptide epitope is capable of introducing an immune response in an animal due to its binding characteristics to MHC molecules. In some embodiments, after recognition of the T cell epitope, such as an MHC peptide complex, TCR (or another MHC peptide-binding molecule) produces or triggers an activation signal for the T cell that it produces a T cell response, such as T cell proliferation, cytokine production, a cytotoxic T cell response, or other response.

[00659] In some embodiments, the TCR, or another MHC peptide binding molecule, recognizes or potentially recognizes the T cell epitope in the context of a class I MHC molecule. Class I MHC proteins are expressed in all nucleated cells of higher vertebrates. The MHC class I molecule is a heterodimer composed of a 46 kDa heavy chain that is non-covalently associated with the 12 kDa light chain β-2 microglobulin. In humans, there are several MHC alleles, such as, for example, HLA-A2, HLA-A1, HLA-A3, HLA-A24, HLA-A28, HLA-A31, HLA-A33, HLA-A34, HLA-B7 , HLA-B45 and HLA-Cw8. The sequences of the MHC alleles are known and can be found, for example, in the IMGT / HLA database available at www.ebi.ac.uk/ipd/imgt/hla. In some modalities, the MHC class I allele is an HLA-A2 allele, which in some populations is expressed by approximately 50% of the population. In some modalities, the HLA-A2 allele may be a product of the HLA-A * 0201, * 0202, * 0203, * 0206, or * 0207 gene. In some cases, there may be differences in the frequency of subtypes between different populations. For example, in some modalities, more than 95% of the HLA-A2 positive Caucusan population is HLA-A * 0201, while in the Chinese population the frequency has been reported to be approximately 23% HLA-A * 0201, 45% HLA-A * 0207, 8% HLA-A * 0206 and 23% HLA-A * 0203.

[00660] In some embodiments, MHC class I restricted peptides are 8 to 15 amino acids in length, such as 8 to 10 amino acids in length. In some embodiments, MHC class I molecules bind to peptides derived from endogenous antigens, such as a tumor, viral or bacterial proteins produced within a diseased or infected cell, which have been processed within the cell's cytoplasm via cytosolic pathway. In some embodiments, MHC class I-peptide complexes displayed on the cell surface are typically recognized by TCRs expressed in CD8 + T cells, such as cytotoxic T cells. In some embodiments, MHC class I peptide complexes can be recognized by TCRs expressed on CD4 + T cells, such as by TCRs exhibiting CD8- or partial CD8- independent binding.

[00661] In some embodiments, the TCR, or another MHC peptide binding molecule, recognizes or potentially recognizes the T cell epitope in the context of a class II MHC molecule. Class II MHC proteins are expressed in a subset of vertebrate nucleated cells, usually called antigen-presenting cells (APCs). In humans, there are

several class II MHC alleles, such as, for example, DR1, DR3, DR4, DR7, DR52, DQ1, DQ2, DQ4, DQ8 and DP1. In some embodiments, the MHC class II allele which is HLA-DRB1 * 0101, an HLA-DRB * 0301, HLA-DRB * 0701, HLA-DRB * 0401 an HLA-DQB1 * 0201. The sequences of the MHC alleles are known and can be found, for example, in the IMGT / HLA database available at www.ebi.ac.uk/ipd/imgt/hla.

[00662] In some embodiments, MHC class II restricted peptides are generally between about 9 and 25 residues long, such as between 15 and 25 residues or 13 and 18 residues long and, in some cases, contain a central binding region of about 9 amino acids or about 12 amino acids. In some modalities, MHC class II molecules bind to peptides derived from exogenous antigens, which are internalized by phagocytosis or endocytosis and processed in the endosomal / lysosomal pathway. In some embodiments, MHC class II peptide complexes displayed on the cell surface are typically recognized by CD4 + cells, such as helper T cells. In some embodiments, the displayed class II MHC peptide complexes can be recognized by TCRs expressed in CD8 + T cells.

[00663] Normally, the peptide epitope or T cell epitope is a peptide portion of an antigen. In some embodiments, the antigen is known, and in some cases, the peptide epitope recognized by the TCR or antigen-binding portion of it (or other MHC peptide-binding molecules) may also be known, as known before performing the method provided.

[00664] In some modalities, the antigen is a tumor-associated antigen, an antigen expressed in a certain type of cell associated with an autoimmune or inflammatory disease, or an antigen derived from a viral or bacterial pathogen. In some modalities, the antigen is an antigen involved in a disease. In some modalities, the disease can be caused by malignancy or cell transformation, such as cancer. In some modalities, the antigen may be an intracellular protein antigen of a tumor or cancer cell, such as a tumor-associated antigen. In some cases, as most cancer antigens are derived from intracellular proteins that can only be directed to the cell surface in the context of an MHC molecule, TCRs are ideal candidates for therapy, as they have evolved to recognize this class of antigens. In some embodiments, the disease can be caused by infection, such as bacterial or viral infection. In some modalities, the antigen is a cancer antigen associated with viruses. In some cases, a recombinant TCR or antigen-binding moieties thereof (and other MHC peptide-binding molecules) recognize or potentially recognize peptides derived from viral proteins that have been naturally processed in infected cells and exhibited by a MHC molecule on the cell surface. In some embodiments, the disease can be an autoimmune disease. Other targets include those listed in The HLA Factsbook (Marsh et al. (2000)) and others that are known.

[00665] In some modalities, the antigen is one that is associated with a tumor or cancer. In some modalities, a tumor or cancer antigen is one that can be found in a malignant cell, found within a malignant cell, or is a mediator of tumor cell growth. In some embodiments, a cancerous tumor or antigen is one that is predominantly expressed or overexpressed by a tumor or cancer cell. A number of tumor antigens have been identified and are known, including tumor antigens defined by T cells restricted to

MHC (see for example, cancerimmunity.org/peptide/; Boon and Old (1997) Curr Opin Immunol, 9: 681-3; Cheever et al. (2009) Clin Cancer Res, 15: 5323-37). In some embodiments, tumor antigens include, but are not limited to, mutated peptides, differentiation antigens and overexpressed antigens, all of which can serve as targets for therapies.

[00666] In some embodiments, the cancer tumor or antigen is a lymphoma antigen, (for example, non-Hodgkin's lymphoma or Hodgkin's lymphoma), a B cell lymphoma cancer antigen, a leukemia antigen, a myeloma antigen (ie, multiple myeloma or plasma cell myeloma), and acute lymphoblastic leukemia antigen, a chronic myeloid leukemia antigen, or an acute myeloid leukemia antigen. In some embodiments, the cancer antigen is an antigen overexpressed in or associated with a cancer that is an adenocarcinoma, such as pancreas, colon, breast, ovary, lung, prostate, head and neck, including multiple myelomas and some cell lymphomas B. In some embodiments, the antigen is associated with cancer, such as prostate cancer, lung cancer, breast cancer, ovarian cancer, pancreatic cancer, skin cancer, liver cancer (for example, adenocarcinoma hepocellular), bowel or bladder cancer.

[00667] In some modalities, the antigen is a tumor antigen that can be an antigen associated with glioma, β-human chorionic gonadotropin, alpha-fetoprotein (AFP), B cell maturation antigen (BCMA, BCM), receptor B cell activation factor (BAFFR, BR3), and / or transmembrane activator and CAML interactor (TACI), Fc 5 receptor (FCRL5, FcRH5), AFP reactive to lectin, thyroglobulin, RAGE-1, MN-CA IX , human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, Methylin-A / MART-1, WT-1, S-100, MBP, CD63, MUC1 (for example,

MUC1-8), p53, Ras, cyclin B1, HER-2 / neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE- A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-C1, BAGE, GAGE- 1, GAGE-2, p5, tyrosinase (e.g., tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, IFN-inducible p78, and melanotransferrin (p97), Uroplakin II, prostate specific antigen (PSA ), human kallikrein (huK2), prostate specific membrane antigen (PSM), prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, beta-catenin, Bcr-abl, E2A-PRL, H4 -RET, IGH-IGK, MYL-RAR, Caspase 8 or a B-Raf antigen. Other tumor antigens can include any derivative of FRa, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22 , ROR1, mesothelin, CD33 / IL3Ra, c- Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin. Specific tumor-associated antigens or T-cell epitopes are known (see, for example, van der Bruggen et al. (2013) Cancer Immun, available at www.cancerimmunity.org/peptide/; Cheever et al. (2009) Clin Cancer Res, 15, 5323-37).

[00668] In some modalities, the antigen is a viral antigen. Many targets of viral antigens have been identified and are known, including peptides derived from viral genomes in HIV, HTLV and other viruses (see, for example, Addo et al. (2007) PLoS ONE, 2, e321; Tsomides et al. (1994) J Exp Med, 180, 1283-93; Utz et al. (1996) J Virol, 70, 843-51). Exemplary viral antigens include, but are not limited to, an antigen from hepatitis A, hepatitis B (eg,

HBV surface and core antigens (HBVc, HBVs), hepatitis C (HCV), Epstein-Barr virus (eg EBVA), human papilloma virus (HPV; eg E6 and E7), hu immunodeficiency virus - type 1 mana (HIV1), herpes Kaposi's sarcoma virus (KSHV), human papilloma virus (HPV), influenza virus, Lassa virus, HTLN- 1, HIN-1, HIN-II, CMN, EBN or HPN . In some embodiments, the target protein is a bacterial antigen or other pathogenic antigen, such as Mycobacterium tuberculosis (MT) antigens, trypanosome, for example, Tiypansoma cruzi (T. cruzi), antigens such as surface antigen (TSA), or malaria antigens. Specific viral antigens or epitopes or other pathogenic antigens or T cell epitopes are known (see, for example, Addo et al. (2007) PLoS ONE, 2: e321; Anikeeva et al. (2009) Clin Immunol, 130: 98- 109).

[00669] In some modalities, the antigen is an antigen derived from a virus associated with cancer, such as an oncogenic virus. For example, an oncogenic virus is one in which the infection of certain viruses is known to lead to the development of different types of cancer, for example, hepatitis A, hepatitis B (for example, HBV surface and core antigens (HBVc , HBVs)), Hepatitis C (HCV), human papilloma virus (HPV), hepatitis viral infections, Epstein-Barr virus (EBV), human herpes virus virus (HHV-8), virus 1 of human T cell leukemia (HTLV-1), human T cell leukemia virus 2 (HTLV-2), or a cytomegalovirus antigen (CMV).

[00670] In some modalities, the viral antigen is an HPV antigen, which, in some cases, can lead to an increased risk of developing cervical cancer. In some embodiments, the antigen may be an HPV-16 antigen, an HPV-18 antigen, an HPV-31 antigen, an HPV-33 antigen or an HPV-35 antigen. In some embodiments, the viral antigen is an HPV-16 antigen (for example, seroreactive regions of the HPV-16 proteins E1, E2, E6 and / or E7,

see, for example, U.S. Patent No. 6,531,127) or an HPV-18 antigen (for example, seroreactive regions of HPV-18 L1 and / or L2 proteins, as described in U.S. Patent No. .

5,840,306). In some embodiments, the viral antigen is an HPV-16 antigen from HPV-16 E6 and / or E7 proteins. In some modalities, the TCR is a TCR directed against an HPV-16 E6 or HPV-16 E7. In some embodiments, the TCR is a TCR described in, for example, WO 2015/184228, WO 2015/009604 and WO 2015/009606.

[00671] In some modalities, the viral antigen is an HBV or HCV antigen, which, in some cases, may lead to a higher risk of developing liver cancer than HBV or HCV negative individuals. For example, in some embodiments, the heterologous antigen is an HBV antigen, such as a central hepatitis B antigen or a hepatitis B envelope antigen (US 2012/0308580).

[00672] In some modalities, the viral antigen is an EBV antigen that, in some cases, may lead to a greater risk of developing Burkitt's lymphoma, nasopharyngeal carcinoma and Hodgkin's disease than EBV-negative individuals. For example, EBV is a human herpes virus that, in some cases, is found associated with numerous human tumors from different tissue origins. Although primarily found as an asymptomatic infection, EBV-positive tumors can be specified by active expression of viral gene products, such as EBNA-1, LMP-1 and LMP-2A. In some embodiments, the heterologous antigen is an EBV antigen that may include the Epstein-Barr nuclear antigen (EBNA) -1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein ( EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA or EBV-VCA.

[00673] In some modalities, the viral antigen is an HTLV-1 or HTLV-2 antigen, which, in some cases, may lead to a greater risk of developing T-cell leukemia than HTLV-1-negative individuals or HTLV-2. For example, in some embodiments, the heterologous antigen is an HTLV antigen, such as TAX.

[00674] In some modalities, the viral antigen is an HHV-8 antigen, which, in some cases, may lead to a greater risk of developing Kaposi's sarcoma than HHV-8 negative individuals. In some embodiments, the heterologous antigen is a CMV antigen, such as pp65 or pp64 (see, U.S. Patent No.

8,361,473).

[00676] In some embodiments, the antigen is a self-antigen, such as a polypeptide antigen associated with an autoimmune disease or disorder. In some modalities, the disease or autoimmune disorder may be multiple sclerosis (MS), rheumatoid arthritis (RA), Sjogren's syndrome, scleroderma, polymyositis, dermatomyositis, systemic lupus erythematosus, juvenile rheumatoid arthritis, severe ankylosing spondylitis, severe mitching (MG), bullous pemphigus (antibodies to the basement membrane at the dermal-epidermal junction), pemphigus (antibodies to the mucopolysaccharide protein complex or intracellular cement substance), glomerulonephritis (antibodies to the glomerular basement membrane), Goodpasture syndrome, autoimmune hemolytic anemia (antibodies to erythrocytes), Hashimoto's disease (antibodies to thyroid), pernicious anemia (antibodies to intrinsic factor), idiopathic thrombocytopenic purpura (antibodies to platelets), Grave's disease, Grave's disease, or Addison's disease ( antibodies to tyro globulin). In some embodiments, the autoantigen, such as an autoantigen associated with an autoimmune disease described in this document, may be collagen, such as collagen type II, mycobacterial heat shock protein, thyroglobulin, acetylcholine receptor ( AcHR), myelin basic protein (MBP) or protolipid protein (PLP). Associated specific autoimmune epitopes or antigens are known (see, for example, Bulek et al. (2012) Nat Immunol, 13: 283-9; Harkiolaki et al. (2009) Immunity, 30: 348-57; Skowera et al. ( 2008) J Clin Invest, 1 (18): 3390-402).

[00677] In some embodiments, the identity of the peptide epitope of the target antigen is known, which, in some cases, can be used in the production or generation of a TCR of interest or in the evaluation of an activity or functional property , including in connection with the methods provided. In some embodiments, peptide epitopes can be determined or identified based on the presence of HLA-restricted motif in a target antigen of interest. In some embodiments, peptides are identified using known computer prediction patterns. In some embodiments, to predict MHC class I binding sites, such standards include, but are not limited to, ProPred1 (Singh and Raghava (2001) Bioinformatics 17 (12): 1236-1237, and SYFPEITHI (see Schuler et (2007) Immuninformatics Methods in Molecular Biology, 409 (1): 75-93 2007) In some embodiments, the MHC-restricted epitope is HLA-A0201, which is expressed in approximately 39 to 46% of all Caucasians and therefore represents an appropriate choice of MHC antigen for use in the preparation of a TCR or other MHC peptide binding molecule In some respects, HLA-A * 0201 binding motifs and cleavage sites for proteasomes and immune proteasomes using computer prediction patterns are known.To predict MHC class I binding sites, these patterns include, but are not limited to, ProPred1 (described in more detail in Singh and Rahava, ProPred: prediction of HLA-DR binding sites, BIOIN-FORMATICS 17 (12): 1236-1237 2001), and SYF PEITHI (see Schuler et al. SYFPEITHI, Database for Searching and T-Cell Epitope Prediction.

in Immunoinformatics Methods in Molecular Biology, vol 409 (1): 75-93 2007). Cell tracking methods used in screening methods, such as T cells, that recognize an antigen or an epitope are provided in the context of a major histocompatibility complex (MHC) molecule.

[00678] In some embodiments, the MHC contains a polymorphic peptide binding site or binding groove that can, in some cases, form complexes with polypeptide peptide epitopes, including peptide epitopes processed by the cellular mechanism. In some cases, MHC molecules can be displayed or expressed on the cell surface, including as a peptide complex, that is, MHC-peptide complex, for presenting an antigen in a TCR-recognizable conformation in T cells, or other binding molecules to MHC-peptide. Generally, MHC class I molecules are heterodimers having a membrane spanning α chain, in some cases with three α domains, and a non-covalently associated β2 microglobulin. Generally, MHC class II molecules are composed of two transmembrane glycoproteins, α and β, both of which normally span the membrane. An MHC molecule can include an effective portion of an MHC that contains an epitope binding site or sites for binding to a peptide and the sequences necessary for recognition by the appropriate binding molecule, such as TCR. In some embodiments, MHC class I molecules release peptides that originate in the cytosol to the cell surface, where a peptide: MHC complex is recognized by T cells, such as generally CD8 + T cells, but in some cases CD4 + T cells. In some modalities, MHC class II molecules release peptides that originate in the vesicular system to the cell surface, where they are normally recognized by CD4 + T cells. Generally, MHC molecules are encoded by a group of linked loci, which are collectively called H-2 in human and mouse leukocyte antigen in humans (HLA). In some ways, human MHC can also be referred to as a human leukocyte antigen (HLA).

[00679] In some embodiments, the peptide epitope or T cell epitope is a peptide that can be derived from or based on a fragment of a longer biological molecule, such as a polypeptide or protein, and which is capable of association with or formation of a complex with an MHC molecule. In some modalities, the peptide is about 8 to about 24 amino acids in length. In some embodiments, the peptide has a length of or about 9 to 22 amino acids for recognition in the MHC Class II complex. In some embodiments, the peptide is about 8 to 13 amino acids long for recognition in the MHC Class I complex. In some embodiments, the MHC molecule and peptide epitope or T cell epitope are combined. plexed or associated through non-covalent interactions of the peptide in the binding groove or slit of the MHC molecule.

[00680] In some embodiments, the MHC-peptide complex is present or is displayed on the surface of cells. In some modalities, the MHC-peptide complex can be specifically recognized by a TCR or antigen-binding portion thereof, or another MHC-peptide-binding molecule. In some embodiments, the T cell epitope or peptide epitope is capable of introducing an immune response in an animal due to its binding characteristics to MHC molecules. In some embodiments, after recognition of the T cell epitope, such as the MHC-peptide complex, a TCR (or other MHC-peptide binding molecule) produces or triggers an activation signal for the T cell that induces a T cell, such as T cell proliferation, cytokine production, a cytotoxic T cell response or other response.

[00681] In some embodiments, the MHC-peptide-binding molecule is a TCR or epitope-binding fragment thereof. In some embodiments, the MHC-peptide-binding molecule is a TCR-like CAR that contains an antibody or epitope-binding fragment thereof, such as a TCR-like antibody, such as one that was designed to bind to complexes MHC-peptides. In some embodiments, such binding molecules bind to a binding sequence, such as a T cell epitope, which contains an amino acid or antigen sequence from a target polypeptide. In some embodiments, the binding sequence of the target peptide or target polypeptide is known. In some embodiments, the MHC-peptide binding molecule can be derived from natural sources, or it can be partially or completely produced synthetically or recombinantly.

[00682] In some embodiments, the MHC-peptide binding molecule is a molecule or part of it that has the ability to bind, for example, specifically to bind, to a peptide epitope that is presented or displayed in the context of an MHC molecule, that is, an MHC-peptide complex, such as on the surface of a cell. In some embodiments, a binding molecule can include any naturally occurring, synthetic, semi-synthetic or recombinantly produced molecule that can bind, for example, specifically bind, to an MHC-peptide complex. Exemplary MHC-peptide binding molecule includes T cell receptors or antibodies, or antigen binding portions thereof, including variable regions of single chain immunoglobulin (eg scTCR, scFv) of the same, that exhibit specific ability to bind to an MHC-peptide complex.

[00683] In some modalities, the TCR is a full-size TCR. In some embodiments, the TCR is an antigen-binding portion. In some embodiments, the TCR is a dimeric TCR (dTCR). In some modalities, the TCR is a simple chain TCR (sc-TCR). A TCR can be attached to the cell or in soluble form. In some embodiments, the TCR is in the form attached to the cell expressed on the surface of a cell.

[00684] In some embodiments, a dTCR contains a first polypeptide in which a sequence corresponding to a supplied TCR α-chain variable region sequence is fused to the N-terminus of a sequence corresponding to an α-chain constant region extracellular sequence TCR, and a second polypeptide in which a sequence corresponding to a sequence of TCR β chain variable region supplied is fused to the N-terminus, a sequence corresponding to an extracellular sequence of TCR β chain constant region , the first and second polypeptides being linked by a disulfide bond. In some embodiments, the bond may correspond to the native interchain disulfide bond present in native dimeric αβ TCRs. In some embodiments, the interchain disulfide bonds are not present in a native TCR. For example, in some embodiments, one or more cysteines can be incorporated into the extracellular sequences of constant region of a dTCR polypeptide pair. In some cases, both native and non-native disulfide bonding may be desirable. In some embodiments, the TCR contains a transmembrane sequence to anchor to the membrane.

[00685] In some embodiments, a dTCR contains a supplied α TCR chain that contains a variable α domain, a constant α domain and a first dimerization motif linked to the C terminal of the constant α domain, and a provided TCR β chain that comprises a variable β domain, a constant β domain and a first dimerization motif attached to the C terminal of the constant β domain, where the first and second dimerization motifs easily interact to form a covalent bond between an amino acid in the first motif of dimerization and an amino acid in the second dimerization motif linking the α chain of TCR and TCR chain together.

[00686] In some embodiments, the TCR is a scTCR, which is a simple amino acid strand that contains an α chain and a β chain that is capable of binding to MHC-peptide complexes. Generally, a scTCR can be generated using known methods, see, for example, PCT published International Nos. WO 96/13593, WO 96/18105, WO 99/18129, WO 04/033685, WO 2006/037960, WO 2011/044186; United States Patent No. 7,569,664; and Schlueter, C. J. et al. J. Mol. Biol. 256, 859 (1996).

[00687] In some embodiments, a scTCR contains a first segment consisting of a sequence of amino acids corresponding to a sequence of a variable region of α chain of TCR provided, a second segment consisting of a sequence of amino acids corresponding to a sequence of variable region of β chain of TCR supplied fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of TCR β-chain constant domain, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[00688] In some embodiments, a scTCR contains a first segment consisting of an amino acid sequence corresponding to a variable region of the β chain of TCR provided, a second segment consisting of an amino acid sequence corresponding to a sequence of a variable region of α chain of TCR supplied fused to the N-terminus of an amino acid sequence corresponding to an extracellular sequence of constant domain of α-chain of TCR, and a linker sequence linking the C-terminus of the first segment to the N-terminus of the second segment.

[00689] In some embodiments, a scTCR contains a first segment consisting of an α chain variable region sequence delivered fused to the N-terminus of an extracellular α chain constant domain sequence, and a second segment consisting of a sequence of α β-chain variable region supplied fused to the N-terminus of an extracellular β-chain constant of transmembrane sequence and sequence, and, optionally, a ligand sequence connecting the C-terminus of the first segment to the N-terminus of the second segment.

[00690] In some embodiments, a scTCR contains a first segment consisting of a sequence of variable region of β chain of TCR supplied fused to the N-terminus of a sequence of extracellular constant domain of chain β, and a second segment constituted by a supplied α-chain variable region sequence fused to the N-terminus of an α-chain extracellular constant of transmembrane sequence and sequence, and, optionally, a linker sequence connecting the C-terminus of the first segment to the N-terminus of the second segment .

[00691] In some embodiments, for the scTCR to bind to an MHC-peptide complex, the α and β chains must be paired so that their variable region sequences are oriented for such a link. Several methods of promoting pairing of α and β in a scTCR are known. In some embodiments, a linker sequence is included that links the α and β chains to form the single polypeptide strand. In some embodiments, the ligand must be of sufficient length to cover the distance between the C terminal of the α chain and the N terminal of the β chain, or vice versa, at the same time, ensuring that the length of the ligand is not so long that it blocks or reduces the binding of scTCR to the target peptide-MHC complex.

[00692] In some embodiments, the scTCRs linker that binds to the first and second TCR segments can be any linker capable of forming a single polypeptide strand, while retaining specific TCR binding. In some embodiments, the linker sequence may, for example, have the formula -P-AA-P-, where P is proline and AA represents an amino acid sequence in which the amino acids are glycine and serine. In some embodiments, the first and second segments are paired so that the variable region sequences of the same are oriented for such a link. Therefore, in some cases, the ligand is long enough to cover the distance between the C terminal of the first segment and the N terminal of the second segment, or vice versa, but it is not so long to block or reduce the connection. scTCR to the target ligand. In some embodiments, the linker may contain either about 10 to 45 amino acids, such as 10 to 30 amino acids or 26 to 41 amino acid residues, for example 29, 30, 31 or 32 amino acids. In some embodiments, the linker has the formula -PGGG- (SGGGG) n-P-, where n is 5 or 6 and P is proline, G is glycine and S is serine (SEQ ID NO: 22). In some modalities, the linker has the sequence GSADDAKKDAAKKDGKS (SEQ ID NO: 23).

[00693] In some embodiments, a scTCR contains a disulfide bond between residues of the simple amino acid strand, which, in some cases, can promote stability of the pairing between the α and β regions of the single chain molecule (see for example , United States Patent No. 7,569,664). In some embodiments, scTCR contains a covalent disulfide bond linking a residue of the immunoglobulin region of the α chain constant domain to a residue of the immunoglobulin region of the β chain constant domain of the single chain molecule. In some embodiments, the disulfide bond corresponds to the native disulfide bond present in a native dTCR. In some embodiments, the disulfide bond in a native TCR is not present. In some embodiments, the disulfide bond is a non-native disulfide bond introduced, for example, incorporating one or more cysteines in the extracellular sequences of constant region of the first and second chain regions of the scTCR polypeptide. Exemplary cysteine mutations include any as described herein. In some cases, both a native and a non-native disulfide bond may be present.

[00694] In some embodiments, a scTCR is a non-disulfide-linked truncated TCR in which heterologous leucine zippers fused to the C-terminal of the same facilitate chain binding (see, for example, PCT published International No. WO 99/60120 ). In some embodiments, a scTCR contains a variable domain of TCRα covalently linked to a variable domain of TCRβ by means of a peptide linker (see, for example, International Published PCT No. WO 99/18129).

[00695] In some embodiments, any of the supplied TCRs, including a dTCR or scTCR, can be linked to signaling domains that produce an active TCR on the surface of a T cell. In some embodiments, the TCR is expressed on the surface of cells. In some embodiments, the TCR contains a sequence corresponding to a transmembrane sequence. In some modes, the transmembrane domain is positively charged. In some embodiments, the transmembrane domain can be a Cα or Cβ transmembrane domain. In some embodiments, the transmembrane domain may be of a non-TCR origin, for example, a transmembrane region of CD3z, CD28 or B7.1. In some embodiments, the TCR contains a sequence corresponding to cytoplasmic sequences. In some modalities, the TCR contains a CD3z signaling domain. In some modalities, the TCR is capable of forming a complex of TCR with CD3.

[00696] In some modalities, the TCR is a soluble TCR. In some embodiments, the soluble TCR has a structure as described in WO 99/60120 or WO 03/020763. In some embodiments, the TCR does not contain a sequence corresponding to the transmembrane sequence, for example, to allow membrane anchoring in the cell in which it is expressed. In some embodiments, the TCR does not contain a sequence corresponding to cytoplasmic sequences.

[00697] In some embodiments, the recombinant receptor, for example, TCR or antigen-binding fragment thereof, is or has been modified in comparison to a known recombinant receptor. In certain embodiments, recombinant receptors, for example, TCRs or antigen-binding fragments thereof, include one or more amino acid variations, for example, substitutions, deletions, insertions, and / or mutations, compared to that of a recombinant receptor, for example, TCR, described in the present document or known. Exemplary variants include those designed to improve the binding affinity and / or other biological properties of the binding molecule. Amino acid sequence variants of a binding molecule can be prepared by introducing appropriate modifications to the nucleotide sequence encoding a binding molecule, or by peptide synthesis. Such modifications include, for example, deletions of, and / or insertions in and / or substitutions of residues within the amino acid sequences of the binding molecule. Any combination of deletion, insertion, and substitution can be done to arrive at the final construction, as long as the final construction has the desired characteristics, for example, antigen binding.

[00698] In some modalities, directed evolution methods are used to generate TCRs with altered properties, such as high affinity for a specific peptide in the context of an MHC molecule. In some modalities, targeted evolution is achieved by display methods including, but not limited to, yeast display (Holler et al. (2003) Nat Immunol, 4, 55-62; Holler et al. (2000) Proc Natl Acad Sci USA, 97, 5387-92), phage display (Li et al. (2005) Nat Biotechnol, 23, 349-54), or T cell display (Chervin et al. (2008) J Immunol Methods, 339 , 175-84). In some modalities, display methods involve engineering, or modifying, a known, primary or reference TCR. For example, in some cases, a reference TCR, like any provided in this document, can be used as a standard for producing mutagenized TCRs in which one or more CDR residues are mutated, and mutants with a unique property. desired alteration, such as high affinity for peptide epitope in the context of an MHC molecule, are selected.

[00699] In certain embodiments, the recombinant receptors, for example, TCRs or antigen-binding fragment thereof, include one or more amino acid substitutions, for example, when compared to a recombinant receptor, for example, TCR, sequence compared to a sequence of a natural repertoire, for example, human repertoire. Sites of interest for substitutional mutagenesis include CDRs, FRs and / or constant regions. Amino acid substitutions can be introduced into a binding molecule of interest and products classified for a desired activity, for example, retained / improved antigen affinity or avidity, reduced immunogenicity, improved half-life, binding or independent CD8 activity, surface expression, pro-

motion of TCR chain matching and / or other improved properties or functions.

[00700] In some embodiments, one or more residues within a CDR of a recombinant receptor, for example, TCR, is / are replaced (s). In some embodiments, the substitution is made to revert a sequence or position in the sequence to a germline sequence, such as a binding molecule sequence found in the germline (for example, human germline), for example, to reduce the likelihood of immunogenicity, for example, after administration to a human subject.

[00701] In certain embodiments, substitutions, insertions, or deletions may occur within one or more CDRs as long as such changes do not substantially reduce the capacity of the recombinant receptor, for example, TCR or antigen-binding fragment thereof, from bind to the antigen. For example, conservative changes (for example, conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made on CDRs. Such changes may, for example, be out of residues from contact with antigen in CDRs. In certain modalities of the variable sequences provided herein, each CDR is either unchanged, or contains no more than one, two or three amino acid substitutions.

[00702] Amino acid sequence insertions include amino- and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, as well as intrasequence insertions of a single or multiple residues. amino acid.

[00703] In some aspects, the TCR or antigen-binding fragment thereof may contain one or more modifications in the α and / or β-chain so that when the TCR or antigen-binding fragment is expressed in a cell , the frequency of incorrect pairing between the α chain and β chain of TCR and an α chain and endogenous TCR chain β is reduced, the expression of the α chain and β chain of TCR is increased, and / or the stability of the chain α and β chain of TCR is increased.

[00704] In some embodiments, the TCR contains one or more non-native cysteine residues to introduce a covalent disulfide bond linking a residue in the immunoglobulin region of the constant domain of the α chain to a residue in the immunoglobulin region of the constant domain β chain. In some embodiments, one or more cysteines can be incorporated into the extracellular sequences of the constant region of the first and second segments of the TCR polypeptide. Exemplary non-limiting modifications to a TCR to introduce non-native cysteine residues are described in the present document (see also, International PCT No. WO 2006/000830 and WO 2006037960). In some cases, both native and non-native disulfide bonding may be desirable. In some embodiments, the TCR or antigen-binding fragment is modified so that the disulfide bond interacts with a native TCR is not present.

[00705] In some embodiments, the transmembrane domain of the TCR constant region can be modified to contain a greater number of hydrophobic residues (see, for example, Haga-Friedman et al. (2012) Journal of Immunology, 188: 5538- 5546). In some modes, the TCR α-chain transmembrane region contains one or more mutations corresponding to S116L, G119V or F120L, with reference to the numbering of a set of Cα established in SEQ ID NO: 24.

[00706] In some embodiments, the TCR or antigen-binding fragment thereof is encoded by a nucleotide sequence that is or has been codon-optimized. Exemplary codon-optimized variants are described elsewhere in this document. V. COMPOSITIONS AND FORMULATIONS

[00707] Projected cell populations, compositions containing such cells and / or enriched for such cells are also provided. Among the compositions are pharmaceutical compositions and formulations for administration, such as for adoptive cell therapy. Methods are also provided to administer the cells and compositions to individuals, for example, patients.

[00708] In some embodiments, the cell population provided and / or compositions containing projected cells include a cell population that exhibits improved, uniform, homogeneous expression and / or stable expression and / or antigen binding by the recombinant receptor, for example, they exhibit a reduced coefficient of variation compared to the expression and / or antigen binding of cell populations and / or compositions generated using conventional methods. In some modalities, the cell population and / or compositions are at least 100%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20% or 10% lower of variation in expression of the transgene and / or binding to the antigen by the recombinant receptor compared to a respective population generated using conventional methods, for example, random transgene integration. The coefficient of variation is defined as the standard deviation of expression of the nucleic acid of interest (for example, transgene encoding a matching receptor) within a cell population, for example, CD4 + and / or CD8 + T cells , divided by the average expression of the respective nucleic acid of interest in the respective cell population. In some modalities, the cell population and / or compositions exhibit a coefficient of variation that is less than 0.70, 0.65, 0.60, 0.55, 0.50, 0.45, 0.40, 0, 35 or 0.30 or less, when measured among populations

CD4 + and / or CD8 + T cell configurations that have been designed using the methods provided in this document.

[00709] In some embodiments, cell populations and / or compositions are provided that include cells that have a targeted breakdown of the transgene encoding recombinant receptor on one or more of the endogenous TCR gene locus, thereby having one in - caution of one or more of the endogenous TCR gene locus, for example, knockout of the target gene for integration, such as TRAC, TRBC1 and / or TRBC2. In some embodiments, all or substantially all cells in the cell population that have integration of the recombinant receptor encoding gene also have a knockout of one or more endogenous TCR gene locus. In some modalities, at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the cells in the cell population and / or composition expressing the recombinant receptor, contain a knockout of one or more of the endogenous TCR gene locus, for example, TRAC, TRBC1 and / or TRBC2. Thus, in cell populations and / or compositions, all or substantially all of the modified cells that express the recombinant receptor, also contain an endogenous TCR knockout, due to the targeted breakdown of the transgene in the endogenous TCR gene locus.

[00710] In some embodiments, cell populations and / or compositions are provided that include a plurality of engineered immune cells comprising a recombinant receptor or an antigen-binding fragment encoded by a transgene and a rupture genetics of at least one target site within a T cell receptor alpha constant gene (TRAC) and / or a T cell receptor constant beta gene (TRBC), in which at least or more than 35 %, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition comprise a genetic disruption

ca at a target position within a T cell receptor alpha constant gene (TRAC) and / or a T cell receptor beta constant gene (TRBC); and the transgene encoding the recombinant TCR or antigen-binding fragment thereof or a chain thereof is directed to or near the target position by means of homology-directed repair (HDR).

[00711] In some embodiments, expression and / or binding to the antigen by the recombinant receptor can be assessed using any reagents and / or assays described in this document, for example, in Section IC. In some embodiments, expression is measured using a binding molecule that recognizes and / or specifically binds to the recombinant receptor or a portion thereof. For example, in some embodiments, expression of the recombinant receptor encoded by the transgene is assessed using an anti-TCR Vβ 22 antibody, for example, by flow cytometry. In some modalities, binding to the antigen of a recombinant receptor that is a TCR, can be evaluated using antigen that is isolated or purified or recombinant, cells expressing a particular antigen, and / or using a TCR ligand (MHC- peptide).

[00712] In some embodiments, the provided compositions containing cells such as cells expressing the recombinant receptor and / or containing a knockout of one or more of the genes encoding endogenous TCR make at least 30%, 40%, 50% , 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more of the total cells in the composition or cells of a certain type such as CD8 + or CD4 + T cells.

[00713] Compositions including cells for administration are also provided, including pharmaceutical compositions and formulations, such as unit dosage form compositions including several cells for administration in a given dose or fraction thereof. Pharmaceutical compositions and formulations generally include one or more optional pharmaceutically acceptable carriers or excipients. In some embodiments, the composition includes at least one additional therapeutic agent.

[00714] The term "pharmaceutical formulation" refers to a preparation that is in such a way as to allow the biological activity of an active ingredient contained therein to be effective, and that it does not contain additional components that are unacceptably toxic to a individual to whom the formulation is to be administered.

[00715] A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is non-toxic to an individual. A pharmaceutically acceptable vehicle includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

[00716] In some respects, the choice of vehicle is determined in part by the particular cell and / or the method of administration. As a result, there are a variety of suitable formulations. For example, the pharmaceutical composition can contain preservatives. Suitable preservatives may include, for example, methylparaben, propylparaben, sodium benzoate, and benzalkonium chloride. In some ways, a mixture of two or more preservatives is used. The preservative or mixtures thereof are typically present in an amount of about 0.0001% to about 2% by weight of the total composition. Vehicles are described, for example, by Remington's Pharmaceutical Sciences Sciences 16th edition, Osol, A. Ed. (1980). Pharmaceutically acceptable vehicles are generally non-toxic to containers at the dosages and concentrations used, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethoxyl chloride

nio; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight polypeptides (less than about 10 residues); proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (for example, Zn-protein complexes); and / or non-ionic surfactants such as polyethylene glycol (PEG).

[00717] Buffering agents in some aspects are included in the compositions. Suitable buffering agents include, for example, citric acid, sodium citrate, phosphoric acid, potassium phosphate, and various other acids and salts. In some ways, a mixture of two or more buffering agents is used. The buffering agent or mixtures thereof are typically present in an amount of about 0.001% to about 4% by weight of the total composition. Methods for preparing administrable pharmaceutical compositions are known. Exemplary methods are described in more detail in, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st ed. (May 1, 2005).

[00718] Formulations can include aqueous solutions. The formulation or composition may also contain more than one active ingredient useful for the particular indication, disease, or condition being treated with the cells, preferably that with complementary activities for the cells, where the respective activities do not affect ad - quite another. Such active ingredients are suitably present in combination in amounts that are effective for the intended purpose. Thus, in some embodiments, the pharmaceutical composition also includes other pharmaceutically active agents or drugs, such as chemotherapeutic agents, for example, asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea, methotrexate , pacli- taxel, rituximab, vinblastine, and / or vincristine.

[00719] The pharmaceutical composition in some embodiments contains the cells in amounts effective to treat or prevent the disease or condition, such as a therapeutically effective or prophylactically effective amount. Therapeutic or prophylactic efficacy in some modalities is monitored by periodic assessment of treated individuals. The desired dosage can be delivered by a single bolus administration of the cells, by multiple bolus administration of the cells, or by administration by continuous infusion of the cells.

[00720] Cells and compositions can be administered using standard administration techniques, formulations, and / or devices. Cell administration can be autologous or heterologous. For example, immunoresponsive or progenitor cells can be obtained from an individual, and administered to the same or a different, compatible individual. Immunoresponsive cells derived from peripheral blood or its progeny (eg, in vivo, ex vivo or derived in vitro) can be administered by localized injection, including catheter administration, systemic injection, localized injection, intravenous injection, or administration parenteral. When administering a therapeutic composition (for example, a pharmaceutical composition that contains a genetically modified immunoresponsive cell), it will usually be formulated in an injectable unit dosage form (solution, suspension, emulsion).

[00721] Formulations include those for oral administration, including

travenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or suppository. In some embodiments, cell populations are administered parenterally. The term "parenteral," as used herein, includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. In some modalities, the cells are administered to the individual using peripheral systemic release by intravenous, intraperitoneal, or subcutaneous injection.

[00722] Compositions in some modalities are provided as sterile liquid preparations, for example, isotonic aqueous solutions, suspensions, emulsions, dispersions, or viscous compositions, which in some respects can be buffered to a selected pH. Liquid preparations are usually easier to prepare than gels, other viscous compositions, and solid compositions. In addition, liquid compositions are a little more convenient to administer, especially by injection. Viscous compositions, on the other hand, can be formulated within the appropriate viscosity range to provide longer contact periods with specific fabrics. Liquid or viscous compositions may comprise vehicles, which may be a solvent or dispersion medium containing, for example, water, saline, phosphate buffered saline, polyol (eg, glycerol, propylene glycol, liquid polyethylene glycol ) and suitable mixtures thereof.

[00723] Sterile injectable solutions can be prepared by incorporating the cells in a solvent, such as in mixture with a suitable vehicle, diluent, or excipient such as sterile water, physiological saline, glucose, dextrose, or the like. The compositions can contain auxiliary substances such as wetting, dispersing, or emulsifying agents (for example, methylcellulose), pH buffering agents, gelling additives or viscosity enhancers

preservatives, flavoring agents, and / or dyes, depending on the route of administration and the desired preparation. Standard texts can in some respects be consulted to prepare suitable preparations.

[00724] Various additives that enhance the stability and sterility of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers, can be added. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, and sorbic acid. Prolonged absorption of the injectable pharmaceutical form can be caused by the use of absorption retarding agents, for example, aluminum monostearate and gelatin.

[00725] The formulations to be used for in vivo administration are generally sterile. Sterility can be readily achieved, for example, by filtration through sterile filtration membranes. SAW. METHODS OF ADMINISTRATION AND USES IN THERAPY

ADOPTIVE MOBILE

[00726] Methods of administering cells, populations, and compositions, and uses of such cells, populations, and compositions to treat or prevent diseases, conditions, and disorders, including cancers, are provided. In some modalities, cells, populations, and compositions are administered to an individual or patient with the particular disease or condition being treated, for example, through adoptive cell therapy, such as T cell therapy. adoptive. In some embodiments, cells and compositions prepared by the methods provided, such as engineered compositions and end-of-production compositions following incubation and / or other processing steps, are administered to an individual, such as an individual having or at risk for the disease or condition. In some ways, the methods,

in this way, they treat, for example, improve one or more symptoms of the disease or condition, such as decreasing the tumor burden in a cancer expressing an antigen recognized by a projected T cell.

[00727] As used herein, an "individual" is a mammal, such as a human or other animal, and is usually human. In some embodiments, the individual, for example, a patient, to whom the cells, cell populations, or compositions are administered is a mammal, usually a primate, such as a human. In some modalities, the primate is either a monkey or an ape. The individual can be male or female and can be at any suitable age, including infant, juvenile, adolescent, adult, and geriatric individuals. In some embodiments, the individual is a non-primate mammal, such as a rodent.

[00728] As used herein, "treatment" (and grammatical variations thereof such as "treating" or "treating") refers to partial or complete improvement or reduction of the disease or condition or disorder, or a symptom adverse effect or result, or associated phenotype. Desirable treatment effects include, but are not limited to, preventing the occurrence or recurrence of disease, relieving symptoms, decreasing any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, improving or palliating disease status, and improved transmission or prognosis. The terms do not imply a complete cure of the disease or complete elimination of any symptom or effect on all symptoms or results.

[00729] As used in this document, "delaying the development of the disease" means postponing, preventing, delaying, delaying, stabilizing, suppressing and / or postponing the development of the disease (such as cancer). This delay can be for several periods of time, depending on the history of the disease and / or the individual treated. A sufficient delay

or significant can, in effect, encompass prevention, in which the individual does not develop the disease. For example, cancer at an advanced stage, such as the development of metastasis, can be delayed.

[00730] "Prevention," as used herein, includes providing prophylaxis with respect to the occurrence or recurrence of the disease in an individual who may be predisposed to the disease, but has not yet been diagnosed with the disease. In some embodiments, the cells provided and compositions are used to slow the development of the disease or to slow the progression of the disease.

[00731] As used in this document, to "suppress" a function or activity is to reduce the function or activity in comparison to the same conditions except for one condition or parameter of interest, or alternatively, in comparison to another condition. For example, cells that suppress tumor growth, reduce the rate of tumor growth compared to the rate of tumor growth in the absence of cells.

[00732] An "effective amount" of a pharmaceutical formulation, cells, or composition, in the context of administration, refers to an effective amount, in dosages / quantities and for periods of time necessary, to achieve a desired result, such as a therapeutic or prophylactic outcome.

[00733] A "therapeutically effective amount" of a pharmaceutical formulation or cells, refers to an effective amount, in dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treating a disease disease, condition, or disorder, and / or pharmacokinetic or pharmacodynamic effect of treatment. The therapeutically effective amount may vary according to factors such as the condition of the disease, age, sex, and weight of the individual, and the populations of cells administered. In some embodiments, the methods provided involve administering the cells and / or compositions in effective amounts, for example, therapeutically effective amounts.

[00734] A "prophylactically effective amount" refers to an effective amount, in dosages and for periods of time necessary, to achieve the desired prophylactic result. Usually, but not necessarily, since a prophylactic dose is used in individuals before or at an early stage of the disease, the prophylactically effective amount will be less than the therapeutically effective amount. In the context of low tumor burden, the prophylactically effective amount in some aspects will be greater than the therapeutically effective amount.

[00735] Methods for administering cells for adoptive cell therapy are known and can be used in conjunction with the methods and compositions provided. For example, methods of adoptive T cell therapy are described, for example, in U.S. Patent Application Publication No. 2003/0170238 to Gruenberg et al; United States Patent No. 4,690,915 to Rosenberg; Rosenberg (2011) Nat Rev Clin Oncol. 8 (10): 577-85). See, for example, Themeli et al. (2013) Nat Biotechnol. 31 (10): 928-933; Tsukahara et al. (2013) Biochem Biophys Res Commun 438 (1): 84-9; Davila et al. (2013) PLoS ONE 8 (4): e61338.

[00736] In some modalities, cell therapy, for example, adoptive T cell therapy, is performed by autologous transfer, in which the cells are isolated and / or otherwise prepared from the individual who should receive the cell therapy, or a sample derived from such an individual. Thus, in some aspects, the cells are derived from an individual, for example, a patient, in need of treatment and the cells, after isolation and processing, are administered to the same individual.

[00737] In some modalities, cell therapy, for example, adoptive T cell therapy, is performed by allogeneic transfer, in which cells are isolated and / or otherwise prepared from an individual other than an individual who should receive or has recently received cell therapy, for example, a first individual. In such modalities, the cells are then administered to a different individual, for example, a second individual, of the same species. In some modalities, the first and second individuals are genetically identical. In some embodiments, the first and second individuals are genetically similar. In some modalities, the second individual expresses the same class of HLA or supertype as the first individual.

[00738] The cells can be administered by any suitable means. Dosage and administration may depend in part, whether administration is brief or chronic. Various dosing schedules include, but are not limited to, single or multiple administrations over various time points, bolus administration, and pulse infusion.

[00739] In some embodiments, the individual was treated with a therapeutic agent targeting the disease or condition, for example, the tumor, prior to administration of the cells or composition containing the cells. In some aspects, the individual is refractory or not responsive to the other therapeutic agent. In some modalities, the individual has persistent or relapsing disease, for example, after treatment with another therapeutic intervention, including chemotherapy, radiation, and / or hematopoietic stem cell transplantation (HSCT), for example, allogeneic HSCT. In some modalities, the administration effectively treats the individual despite the fact that the individual has become resistant to another therapy.

[00740] In some modalities, the individual is responsive to the other therapeutic agent, and treatment with the therapeutic agent reduces disease burden. In some aspects, the individual is initially responsible for the therapeutic agent, but exhibits a recurrence of the disease or condition over time. In some modalities, the individual did not relapse. In some such modalities, the individual is determined to be at risk of relapse, as well as at high risk of relapse, and thus the cells are administered prophylactically, for example, to reduce the likelihood of or preventing relapse.

[00741] In some aspects, the individual did not relapse before treatment with another therapeutic agent.

[00742] The disease or condition that is treated in some aspects may be any one in which expression of an antigen is associated with, specific to, and / or expressed in a cell or tissue of the disease, disorder or condition and / or involved in the etiology of the disease, condition or disorder, for example, cause, exacerbation or otherwise is involved in such a disease, condition, or disorder. Exemplary diseases and conditions can include diseases or conditions associated with malignancy or cell transformation (for example, cancer), autoimmune or inflammatory disease, or an infectious disease, for example, caused by a bacterium, virus, or other pathogen. Exemplary antigens, which include antigens associated with various diseases and conditions that can be treated, are described in this document. In particular embodiments, the immunomodulatory polypeptide and / or recombinant receptor, for example, the chimeric antigen receptor or TCR, specifically binds to an antigen associated with the disease or condition. In some embodiments, the individual has a disease, disorder or condition, optionally a cancer, a tumor, an autoimmune disease, disorder or condition, or an infectious disease.

[00743] In some embodiments, the disease, disorder or condition includes tumors associated with various cancers. Cancer in some ways may be any cancer located in an individual's body, such as, but not limited to, cancers located in the head and neck, breast, liver, colon, ovary, prostate, pancreas, brain , cervix, bone, skin, eye, bladder, stomach, esophagus, peritoneum, or lung. For example, the anticancer agent can be used for the treatment of colon cancer, cervical cancer, cancer of the central nervous system, breast cancer, bladder cancer, anal carcinoma, head and neck cancer, ovarian cancer, endometrial cancer , small cell lung cancer, non-small cell lung cancer, neuroendocrine cancer, soft tissue carcinoma, penile cancer, prostate cancer, pancreatic cancer, gastric cancer, gallbladder cancer or esophageal cancer. In some cases, the cancer can be a blood cancer. In some embodiments, the disease, disorder or condition is a tumor, such as a solid tumor, lymphoma, leukemia, blood tumor, metastatic tumor, or other cancer or type of tumor. In some modalities, the disease, disorder or condition is selected from cancers of the colon, lung, liver, breast, prostate, ovary, skin, melanoma, bone, brain cancer, ovarian cancer, epithelial cancers, renal cell carcinoma, pancreatic adenocarcinoma , cervical carcinoma, colorectal cancer, glioblastoma, neuroblastoma, Ewing's sarcoma, medulloblastoma, osteosarcoma, synovial sarcoma, and / or mesothelioma.

[00744] Among the diseases, conditions and disorders are tumors, including solid tumors, hematological malignancies, and melanomas, and including localized tumors and metastatic infectious diseases, such as an infection with a virus or other pathogen, for example, HIV, HCV , HBV, CMV, HPV, and parasitic disease, and autoimmune and inflammatory diseases. In some modalities, the disease, disorder or condition is a tumor, cancer, malignancy, neoplasm, or other disease

or proliferative disorder. Such diseases include but are not limited to leukemia, lymphoma, for example, acute myeloid leukemia (AML) or (myelogenous), chronic myeloid (or myelogenous) leukemia (CML), acute lymphocytic (or lymphoblastic) leukemia (ALL), chronic lymphocytic leukemia (CLL), hair cell leukemia (HCL), small lymphocytic lymphoma (SLL), mantle cell lymphoma (MCL), marginal zone lymphoma, Burkitt's lymphoma, Hodgkin's lymphoma (HL), Non-Hodgkin's lymphoma (NHL), Anaplastic large cell lymphoma (ALCL), Follicular lymphoma, Refractory follicular lymphoma, Diffuse large B-cell lymphoma (DLBCL) and multiple myeloma (MM), a B cell malignancy is selected from acute lymphoblastic leukemia (ALL), adult ALL, chronic lymphoblastic leukemia (CLL), non-Hodgkin's lymphoma (NHL), and large diffuse B-cell lymphoma (DLBCL).

[00745] In some embodiments, a disease or condition is an infectious disease or condition, such as, but not limited to, viral, retroviral, bacterial, and protozoal infections, immunodeficiency, Cytomegalovirus (CMV), Epstein-Barr Virus ( EBV), adenovirus, Poliomavirus BK. In some embodiments, a disease or condition is an autoimmune or inflammatory disease or condition, such as arthritis, for example, rheumatoid arthritis (RA), Type I Diabetes, systemic lupus erythematosus (SLE), inflammatory bowel disease, psoriasis, scleroderma, autoimmune thyroid disease, Grave's disease, Crohn's disease, multiple sclerosis, asthma, and / or a disease or condition associated with transplantation.

[00746] In some embodiments, the antigen associated with the disease, disorder or condition is selected from ROR1, B cell maturation antigen (BCMA), carbonic anhydrase (CAIX), tEGFR, Her2 / neu (erbB2 receptor tyrosine kinase) , L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and hepatitis B surface antigen, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, glyco-

epithelial protein 2 (EPG-2), epithelial glycoprotein 40 (EPG-40), EPHa2, erb-B2, erb-B3, erb-B4, erbB, EGFR vIII, folate-binding protein (FBP) , FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kinase insertion domain receptor (kdr), kappa light chain, Lewis Y, molecule cell adhesion L1, (L1-CAM), Melanoma-associated antigen (MAGE) -A1, MAGE-A3, MAGE-A6, Preferably expressed melanoma antigen (PRAME), survivin, TAG72, B7-H6, alpha 2 receptor of IL-13 (IL-13Ra2), CA9, GD3, HMW-MAA, CD171, G250 / CAIX, HLA-AI MAGE A1, HLA-A2 NY-ESO-1, PSCA, folate receptor, CD44v6, CD44v7 / 8, integrin avb6, 8H9, NCAM, VEGF receptors, 5T4, fetal AchR, NKG2D, CD44v6 ligands, dual antigen, a mesotheline testicular cancer antigen, murine CMV, mucin 1 (MUC1), MUC16, PSCA, NKG2D, NY -ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), Her2 / neu, estrogen receptor o, progesterone receptor, ephrinB2, CD123, c-Met, GD-2, O-acetylated GD2 (OGD2), CE7, Wilms' Tumor 1 (WT-1), a cyclin, cyclin A2, CCL-1, CD138, a specific pathogen antigen.

[00747] In some embodiments, the antigen associated with a disease or disorder is selected from the group consisting of orphan tyrosine kinase receptor ROR1, tEGFR, Her2, L1-CAM, CD19, CD20, CD22, mesothelin, CEA, and antigen from hepatitis B surface, antifolate receptor, CD23, CD24, CD30, CD33, CD38, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, 3, or 4, FBP, acetylcholine and receptor, GD2, GD3 , HMW-MAA, IL-22R-alpha, IL-13R-alpha2, kdr, kappa light chain, Lewis Y, L1 cell adhesion molecule, MAGE-A1, mesothelin, MUC1, MUC16, PSCA, NKG2D, NY- ESO-1, MART-1, gp100, oncofetal antigen, ROR1, TAG72, VEGF-R2, carcinoembryonic antigen (CEA), prostate specific antigen, PSMA, Her2 / neu, recept-

estrogen receptor, progesterone receptor, ephrinB2, CD123, CS-1, c-Met, GD-2, and MAGE A3 and / or biotinylated molecules, and / or molecules expressed by HIV, HCV, HBV or other pathogens.

[00748] In some embodiments, the cells are administered in a desired dosage, which in some aspects includes a desired dose or number of cells or cell types and / or a desired ratio of cell types. Thus, the dosage of cells in some modalities is based on a total number of cells (or number per kg of body weight) and a desired ratio of the individual populations or subtypes, such as the ratio of CD4 + for CD8 +. In some embodiments, cell dosage is based on a desired total number (or number per kg of body weight) of cells in individual populations or individual cell types. In some embodiments, the dosage is based on a combination of such aspects, such as a desired number of total cells, desired ratio, and total desired number of cells in individual populations.

[00749] In some embodiments, cell populations or subtypes, such as CD8 + and CD4 + T cells, are administered at or within a tolerated difference from a desired dose of total cells, such as a desired dose of T cells. aspects, the desired dose is a desired number of cells or a desired number of cells per unit of body weight of the individual to whom the cells are administered, for example, cells / kg. In some aspects, the desired dose is, or about a minimum number of cells or minimum number per unit of body weight. In some aspects, among the total cells, administered at the desired dose, individual populations or subtypes are present at or close to a desired productivity ratio (such as the CD4 + to CD8 + ratio), for example, within a given foolish difference

or error of such a relationship.

[00750] In some embodiments, cells are administered in or within a tolerated difference in a desired dose of one or more of the individual populations or subtypes of cells, such as a desired dose of CD4 + cells and / or a desired dose of cell them CD8 +. In some respects, the desired dose is a desired number of cells or subtype or population, or desired number of such cells per unit of body weight of the individual to whom the cells are administered, for example, cells / kg. In some respects, the desired dose is at or above a minimum number of cells in the population or subtype, or minimum number of cells in the population or subtype per unit of body weight.

[00751] Thus, in some modalities, the dosage is based on a desired fixed dose of total cells and a desired ratio, and / or based on a fixed dose of one or more, for example, each, of the individual subtypes or subpopulations. Thus, in some modalities, the dosage is based on a desired fixed or minimum dose of T cells and a desired ratio of CD4 + to CD8 + cells, and / or is based on a desired fixed or minimum dose of CD4 + cells. and / or CD8 +.

[00752] In certain modalities, the cells, or individual populations of cell subtypes, are administered to the individual in a range of about one million to about 100 billion cells, such as, for example, 1 million about 50 billion cells (for example, about 5 million cells, about 25 million cells, about 500 million cells, about 1 billion cells, about 5 billion cells, about 20 billion cells, about 30 billion cells, about 40 billion cells, or a range defined by any two of the previous values), such as about 10 million to about 100 billion cells (for example, about 20 million cells, about 30 million cells, about 40 million cells, about 60 million cells, about 70 million cells, about 80 million cells, about 90 million of cells, about 10 billion cells, about 25 billion cells, about 50 billion cells , about 75 billion cells, about 90 billion cells, or a range defined by any two of the previous values), and in some cases about 100 million cells to about 50 billion cells (for example, about 120 million cells, about 250 million cells, about 350 million cells, about 450 million cells, about 650 million cells, about 800 million cells, about 900 million cells, about 3 billion cells, about 30 billion cells, about 45 billion cells) or any value between these ranges.

[00753] In some embodiments, the dose of total cells and / or of individual subpopulations of cells is included in a range between or about 104 and about or about 109 cell / kg (kg) of body weight, such as between 105 and 106 cells / kg of body weight, for example, of or about 1 x 105 cells / kg, 1.5 x 105 cells / kg, 2 x 105 cells / kg, or 1 x 106 cells / kg body weight. For example, in some embodiments, cells are administered at, or within a certain error range of, between or about 104 and about or about 109 T cells / kg (kg) of body weight, such as between 105 and 106 T cells / kg body weight, for example, of or about 1 x 105 T cells / kg, 1.5 x 105 T cells / kg, 2 x 105 T cells / kg, or 1 x 106 T cells / kg body weight.

[00754] In some embodiments, cells are administered at or within a certain error range of between or about 104 and about or about 109 CD4 + and / or CD8 + cells / kg (kg) of body weight, such as between 105 and 106 CD4 + and / or CD8 + cells / kg body weight,

for example, from or about 1 x 105 CD4 + and / or CD8 + cells / kg, 1.5 x 105 CD4 + and / or CD8 + cells / kg, 2 x 105 CD4 + and / or CD8 + cells / kg, or 1 x 106 CD4 + and / or CD8 + cells / kg body weight. +.

[00755] In some embodiments, cells are administered at or within a certain error range of, greater than, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 106 CD4 + cells, and / or at least about 1 x 106, about 2.5 x 106, about 5 x 106, about 7.5 x 106, or about 9 x 10 6 CD8 + cells, and / or at least about 1 x 10 6, about 2.5 x 10 6, about 5 x 10 6, about 7.5 x 10 6, or about 9 x 10 6 T cells . In some embodiments, cells are administered in or within a certain error range of between about 108 and 1012 or between about 1010 and 1011 T cells, between about 108 and 1012 or between about 1010 and 1011 CD4 + cells, and / or + 108 and 1012 or between about 1010 and 1011 CD8 + cells.

[00756] In some embodiments, cells are administered in or within a tolerated range of a desired output ratio of multiple cell populations or subtypes, such as CD4 + and CD8 + cells or subtypes. In some ways, the desired relationship can be a specific relationship or it can be a range of relationships. For example, in some embodiments, the desired ratio (for example, ratio of CD4 + to CD8 + cells) is between or about 1: 5 and about or about 5: 1 (or greater than about 1: 5 and less than than about 5: 1), or between or about 1: 3 and about or about 3: 1 (or greater than about 1: 3 and less than about 3: 1), such as between or about 2: 1 and about or about 1: 5 (or greater than about 1: 5 and less than about 2: 1, such as or about 5: 1, 4.5: 1, 4: 1,

3.5: 1, 3: 1, 2.5: 1, 2: 1, 1.9: 1, 1.8: 1, 1.7: 1, 1.6: 1, 1.5: 1, 1.4: 1, 1.3: 1, 1.2: 1,

1.1: 1, 1: 1, 1: 1.1, 1: 1.2, 1: 1.3, 1: 1.4, 1: 1.5, 1: 1.6, 1: 1.7, 1: 1.8, 1: 1.9: 1: 2, 1: 2.5, 1: 3, 1: 3.5, 1: 4, 1: 4.5, or 1: 5. In some respects, the tolerated difference is within about 1%, about 2%, about 3%, about 4% about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50% of the desired ratio, including any value between these ranges.

[00757] For disease prevention or treatment, the appropriate dosage may depend on the type of disease to be treated, the type of recombinant cells or receptors, the severity and course of the disease, whether the cells are administered for preventive or therapeutic purposes. medications, previous therapies, the individual's medical history and response to cells, and the discretion of the attending physician. The compositions and cells are in some modalities adequately administered to the individual at a time or during a series of treatments.

[00758] In some embodiments, for example, where the individual is a human, the dose includes less than about 1 x 108 cells expressing total recombinant receptor (eg, TCR), T cells, or mononuclear blood cells peripheral (PBMCs), for example, in the range of about 1 x 106 to 1 x 108 of such cells, such as 2 x 106, 5 x 106, 1 x 107, 5 x 107, or 1 x 108 or total of such cells, or the range between any two of the previous values.

[00759] In some respects, the dose size is determined based on one or more criteria such as the individual's response to previous treatment, for example, chemotherapy, disease burden on the individual, such as tumor burden, volume, size or degree, extent, or type of metastasis, stage, and / or probability or incidence of the individual developing toxic results, for example, CRS, macrophage activation syndrome, tumor lysis syndrome, neurotoxicity, and / or an immune response host against the recombinant cells and / or recipients being administered.

[00760] In some respects, the dose size is determined by

the burden of a disease or condition on the individual. For example, in some respects, the number of cells administered in the dose is determined based on the tumor load that is present in the individual immediately before administration of the start of the cell dose. In some modalities, the size of the first and / or subsequent dose is inversely correlated with the disease burden. In some aspects, such as in the context of a high disease burden, the individual is given a low number of cells. In other modalities, such as in the context of a lower disease burden, the individual is given a greater number of cells.

[00761] The cells can be administered by any suitable means, for example, by bolus infusion, by injection, for example, intravenous or subcutaneous injections, intraocular injection, periocular injection, subretinal injection, intravitreal injection, trans-septal injection , subscleral injection, intracoroidal injection, intracameral injection, subconjectival injection, subconjunctival injection, subtendon injection, retrobulbar injection, peribulbar injection, or posterior juxta-scleral release. In some modalities, they are administered by parenteral, intrapulmonary, and intranasal treatment, and, if desired by local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. In some embodiments, a given dose is administered by a single bolus administration of the cells. In some embodiments, it is administered by multiple bolus administrations of the cells, for example, over a period not exceeding 3 days, or by administration of continuous infusion of the cells.

[00762] In some embodiments, cells are administered as part of a combination treatment, such as simultaneously or sequentially with, in any order, another therapeutic intervention, such as an antibody or modified or re- cell.

receptor or agent, such as a cytotoxic or therapeutic agent. Cells in some modalities are co-administered with one or more additional therapeutic agents or in connection with another therapeutic intervention, simultaneously or sequentially in any order. In some contexts, cells are co-administered with another therapy sufficiently close in time, so that cell populations enhance the effect of one or more additional therapeutic agents, or vice versa. In some embodiments, the cells are administered before one or more additional therapeutic agents. In some embodiments, the cells are administered after said one or more additional therapeutic agents. In some embodiments, said one or more additional agents include a cytokine, such as IL-2, for example, to enhance persistence. In some modalities, the methods comprise the administration of a chemotherapeutic agent.

[00763] After administration of the cells, the biological activity of the cell populations modified in some modalities is measured, for example, by any of the several known methods. Evaluation parameters include specific binding of a modified or natural T cell or another cell immune to the antigen, in vivo, for example, by imaging, or ex vivo, for example, by ELISA or flow cytometry. In certain embodiments, the ability of the modified cells to destroy target cells can be measured using any suitable known method, such as cytotoxicity assays described, for example, in Kochenderfer et al., J. Immunotherapy, 32 (7): 689- 702 (2009), and Herman et al. J. Immunological Methods, 285 (1): 25-40 (2004). In certain embodiments, the biological activity of cells is measured by assaying the expression and / or secretion of one or more cytokines, such as CD 107a, IFNγ, IL-2, and TNF. In some aspects, biological activity is measured by assessing the clinical outcome, such as reducing

load or tumor weight.

[00764] In certain modalities, the modified cells are also modified in any number of ways, so that their therapeutic or prophylactic efficacy is increased. For example, the modified CAR or TCR expressed by the population can be conjugated directly or indirectly through a linker to a targeting part. The practice of conjugating compounds, for example, CAR or TCR, to target portions is known. See, for example, Wadwa et al., J. Drug Targeting 3: 1111 (1995), and U.S. Patent No. 5,087,616. VII. KITS and MANUFACTURING ITEMS

[00765] Manufacture articles, systems, apparatus, and kits useful in carrying out the modalities provided are also provided. In some embodiments, manufactured articles or kits contain one or more components of said one or more agents capable of inducing genetic disruption and / or standard polynucleotides, for example, standard polynucleotides containing transgene encoding a recombinant receptor or antigen-binding fragment or chain thereof or said one or more second standard polynucleotides. In some embodiments, articles of manufacture or kits can be used in methods for modifying T cells that express a recombinant receptor and / or other polypeptides and evaluating the cells produced and / or cell populations according to methods provided. In some embodiments, the manufacturing articles or kits provided in this document contain T cells and / or T cell compositions, such as any T cells and / or T cell compositions described in this document.

[00766] In some embodiments, articles of manufacture or kits include polypeptides, nucleic acids, vectors and / or polynucleotides useful in carrying out the methods provided. In some modalities,

articles of manufacture or kits include one or more nucleic acid molecules, for example, a plasmid or a DNA fragment, comprising one or more components of said one or more agents capable of inducing genetic disruption and / or standard polynucleotides , for example, standard polynucleotides containing transgene encoding a recombinant receptor or antigen-binding fragment or chain thereof or said one or more second standard polynucleotides. In some embodiments, the manufacturing articles or kits provided in this document contain control vectors.

[00767] In some embodiments, the articles of manufacture or kits provided in this document contain one or more agents, wherein each of said one or more agents is independently capable of introducing a genetic disruption of a target site within a constant gene cell receptor alpha (TRAC) and / or a T cell receptor beta constant gene (TRBC); and a standard polynucleotide comprising the transgene encoding a recombinant TCR or antigen-binding fragment or strand thereof, wherein the transgene encoding the recombinant TCR or antigen-binding fragment is targeted to integration at or near the target site by means of a targeted repair (HDR).

[00768] In some embodiments, the articles of manufacture or kits provided herein contain T cells, and / or T cell compositions, such as any T cells, and / or T cell compositions described herein. In some embodiments, T cells, and / or T cell compositions, any of the modified T cells used the evaluation methods described herein. In some embodiments, the manufacturing articles or kits provided herein contain control T cells, and / or T cell compositions.

[00769] In some embodiments, articles of manufacture or kits include one or more components used to assess the properties of the population and / or composition of modified cells expressing a recombinant receptor. For example, articles of manufacture or kits may include binding reagents, for example antibodies, MHC peptide tetramers and / or probes, used to evaluate particular properties of the recombinant TCRs introduced, for example, expression of the TCR cell surface recombinant, recognition of a peptide in the context of an MHC molecule and / or detectable signal produced by the reporter, for example, a T cell activation retorter. In some embodiments, manufacturing articles or kits may include components that they are used to detect particular properties, such as labeled components, for example, fluorescently labeled components and / or components that can produce a detectable signal, for example, substrates that can produce fluorescence or luminescence.

[00770] In some embodiments, articles of manufacture or kits include one or more containers, typically a plurality of containers, packaging material, and a label or package leaflet about or associated with the container or containers and / or package, generally including instructions for use, for example, instructions for introducing components into cells for modification and / or for evaluating populations and / or compositions of modified cells. In some embodiments, the article of manufacture or kits includes one or more instructions for administering the modified cells and / or cellular compositions for therapy.

[00771] The manufacturing articles provided in this document contain packaging materials. Packaging materials for use in packaging the supplied materials are known. See, for example, US Patent Nos. 5,323,907,

5,052,558 and 5,033,252, each of which is incorporated herein in its entirety. Examples of packaging materials include, but are not limited to, blister packs, flasks, tubes, inhalers, pumps, bags, vials, containers, syringes, disposable laboratory supplies, for example, pipette tips and / or plastic plates, or bottles. Manufacturing articles or kits may include a device to facilitate the dispensing of materials or to facilitate use in a high-performance or large-scale manner, for example, to facilitate use in robotic equipment. Typically, the packaging is non-reactive with the compositions contained therein.

[00772] In some embodiments, the agent capable of inducing genetic disruption and / or standard polynucleotides are packaged separately. In some embodiments, each container may have a single compartment. In some embodiments, other components of the manufacturing articles or kits are packaged separately, or together in a single compartment. VIII. Definitions

[00773] Unless otherwise defined, all technical and scientific terms, notations and other technical and scientific terms or terminology used herein are intended to have the same meaning as is commonly understood by someone skilled in the art. to which the claimed subject matter belongs. In some cases, terms with the meanings commonly understood in this document are defined for clarity and / or for reference for ready reference, and the inclusion of such definitions in this document should not necessarily be constructed to represent a substantial difference over the which is generally understood.

[00774] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer amino acid residue, and are not limited to a minimum length. Polypeptides, including the supplied antibodies and antibody chains and other peptides, for example, linkers, can include amino acid residues including natural and / or unnatural amino acid residues. The terms also include post-modifications of polypeptide expression, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some respects, polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein retains the desired activity. These modifications may be deliberate, co may be deliberate, such as by means of site-directed mutagenesis, or they may be accidental, such as by means of host mutations that produce proteins or errors due to PCR amplification.

[00775] An "isolated" nucleic acid refers to a nucleic acid molecule that has been separated from a component of its natural environment. A nucleic acid includes a nucleic acid molecule contained in cells that normally contain the nucleic acid molecule, but the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its chromosomal location Natural.

[00776] "Isolated nucleic acid encoding a TCR or an antibody" refers to one or more nucleic acid molecules encoding TCRα or β chains (or fragments thereof) or heavy and light chains of antibody (or fragments thereof), including such nucleic acid molecules in a single vector or separate vectors, and such nucleic acid molecules present at one or more locations in a host cell.

[00777] The terms "host cell," "host cell lineage," and "host cell culture" are used interchangeably and refer to cells in which exogenous nucleic acid has been introduced,

including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the transformed primary cell and progeny derived from them without considering the number of passages. The progeny may not be completely identical in nucleic acid content to a source cell, but may contain mutations. The mutant progeny that has the same biological function or activity as analyzed or selected in the originally transformed cell is included in this document.

[00778] In some embodiments, "operably linked" may include the association of components, such as a DNA sequence, for example, a [heterologous nucleic acid) and regulatory sequences, in a way that allows gene expression when the appropriate molecules (for example, transcriptional activator proteins) are linked to the regulatory sequence. Therefore, it means that the described components are in a connection allowed them to work in their intended way.

[00779] As used herein, "percent (%) amino acid sequence identity" and "percent identity" when used with respect to an amino acid sequence (reference polypeptide sequence) are defined as the percentage of amino acid residues in a candidate sequence (for example, the antibody or object fragment) that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing intervals, if necessary , to obtain the maximum percentage of sequence identity, and not considering any conservative substitutions as part of a sequence identity. Alignment for purposes of determining percentage of amino acid sequence identity can be obtained in several ways that are included in the experiment, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign ( DNASTAR). Appropriate parameters for sequence alignment can be determined, including any algorithms necessary to obtain maximum alignment in the natural size of the sequences being compared.

[00780] An amino acid substitution may include replacing an amino acid in a polypeptide with another amino acid. The substitution can be a conservative amino acid substitution or a non-conservative amino acid substitution. Amino acid substitutions can be introduced into a binding molecule, for example, antibody of interest and the products analyzed for a desired activity, for example, retained / improved antigen binding, reduced immunogenicity, or ADCC or CDC improves - gives.

[00781] Amino acids can generally be grouped according to the following common side chain properties: (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues that influence the chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe.

[00782] In some modalities, conservative substitutions may involve exchanging a member of one of these classes for another member of the same class. In some embodiments, non-conservative amino acid substitutions may involve exchanging a member of these classes for another class.

[00783] The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is attached. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to in this document as "expression vectors."

[00784] The term "package supplement" is used to refer to instructions normally included in commercial therapeutic product packages, which contain information on indications, use, dosage, administration, combination therapy, contraindications and / or warnings about the use of such therapeutic products.

[00785] As used herein, the singular forms "one, one (a)," "one, one (an)," and "o, a (the)" include plural referenttes, unless the context clearly dictates otherwise. For example, "one one (a)" or "one one (an)" means "at least one" or "one or more." It is understood that aspects and variations described in this document include "consisting" and / or "consisting essentially of" aspects and variations.

[00786] Throughout this description, various aspects of the subject matter are presented in a range of formats. It should be understood that the description in a format range is merely for convenience and brevity and should be constructed as an inflexible limitation on the scope of the claimed object matter. Consequently, the description of a range should be considered to have specifically described all possible sub-ranges, as well as individual numerical values within that range. For example, where a range of volumes is provided, it is understood that each intermediate value, between the upper and lower limit of that range and any other established or intermediate value in this established range is covered within the subject matter claimed. The upper and lower limits of these smaller bands can independently be included in the smaller bands, and are also covered within the subject matter claimed, subject to any limit specifically excluded in the established band. Where the established range includes one or both of the limits, ranges including either of the two or both of the included limits are also included in the claimed subject matter. This applies regardless of the range of the range.

[00787] The term "about" as used in this document refers to the usual error range for the respective value easily known by the person skilled in this technical field. Reference to "about" a value or parameter in this document includes (and describes) modalities that target that value or parameter per se. For example, description referring to "about X" includes description of "X". In some embodiments, "about" may refer to ± 25%, ± 20%, ± 15%, ± 10%, ± 5%, or ± 1%.

[00788] As used herein, the composition refers to any mixture of two or more products, substances, or compounds, including cells. It can be a solution or suspension, liquid, powder, a paste, aqueous or non-aqueous or any combination thereof.

[00789] As used herein, a statement that a cell or population of cells is "positive" for a particular marker refers to the detectable presence on or in the cell of a particular marker, typically a surface marker. When referring to a surface marker, the wilderness refers to the presence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting said antibody, in which the The stain is detectable by flow cytometry at a level substantially above the stain detected by performing the same procedure.

assignment with an isotype-compatible control under otherwise identical conditions and / or at a level substantially similar to that for a known cell to be positive for the marker, and / or at a level substantially greater than that for a known cell to be negative for the marker.

[00790] As used herein, an assertion that a cell or cell population is "negative" for a particular marker refers to the absence of substantial detectable presence on or in the cell of a particular marker, typically a mark - surface pain. When referring to a surface marker, the term refers to the absence of surface expression as detected by flow cytometry, for example, by staining with an antibody that specifically binds to the marker and detecting that antibody , in which the staining is not detected by flow cytometry at a level substantially above the detected staining by performing the same procedure with a control compatible with the isotype under conditions of another identical, and / or at a substantially lower level than that for the known cell to be positive for the marker, and / or at a substantially similar level when compared to that for a known cell to be negative for the marker.

[00791] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference . If a definition mentioned in this document is contrary to, or otherwise inconsistent with, a definition mentioned in patents, applications, published applications and other publications that are incorporated in this document by reference, the definition mentioned in this document prevails in the definition that is incorporated in this document by reference.

[00792] The section titles used in this document are for organizational purposes only and should not be construed as limiting the subject matter described IX. EXEMPLARY MODALITIES

[00793] Among the modalities provided are:

[00794] 1. A genetically modified T cell, comprising a modified modified T cell receptor (TRAC) alpha locus, said modified TRAC locus comprising a nucleic acid encoding a recombinant TCR or part thereof, the said recombinant TCR or part thereof comprising: (i) an alpha TCR chain (TCRα) comprising a variable alpha domain (Vα) and a constant domain, and (ii) a beta TCR chain (TCRβ) which comprises comprises a beta variable domain (Vβ) and a constant domain, where the nucleic acid sequence comprises (a) a transgene sequence encoded by the TCRβ and the Vα domain, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, wherein the open reading frame encodes at least a part of the Cα domain of the recombinant TCR.

[00795] 2. The genetically modified T cell of modality 1, in which the transgene sequence is integrated by means of homology-directed repair (HDR).

[00796] 3. The genetically modified T cell of modality 1 or modality 2, in which the modified TRAC locus comprises a fusion in the structure of (i) a transgene sequence and (ii) and an open reading structure or a partial sequence thereof from the endogenous TRAC locus.

[00797] 4. The genetically modified T cell of any one of modalities 1 to 3, in which the transgene sequence does not

it comprises a sequence encoding a 3 'untranslated region (3' UTR) or an intron.

[00798] 5. The genetically modified T cell of any of the modalities 1 to 4, wherein the open reading frame or a partial sequence thereof comprises the 3 'RTU of the endogenous TRAC locus.

[00799] 6. The genetically modified T cell of any of the modalities 1 to 5, in which a part of the Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of the Cα is encoded by the transgene sequence, in which the said other part of Cα is smaller than the natural size of a native Cα.

[00800] 7. The genetically modified T cell of any of the modalities 1 to 6, in which the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus.

[00801] 8. The genetically modified T cell of any of the modalities 1 to 7, in which the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the TRAC locus endogenous.

[00802] 9. The genetically modified T cell of any of modalities 1 to 8, in which the transgene sequence is integrated downstream of the nucleotide plus 5 'of exon 1 and upstream of the nucleotide plus 3' of exon 1 of the open reading structure of the endogenous TRAC locus.

[00803] 10. The genetically modified T cell of any of modalities 1 to 9, wherein said at least a part of Cα is encoded by at least exons 2-4 of the open reading frame of the endogenous TRAC locus.

[00804] 11. The genetically modified T cell of any of the modalities 1 to 10, in which said at least one Cα part is encoded by at least one part of exon 1 and exons 2 to 4 of the open reading frame of the endogenous TRAC locus.

[00805] 12. The genetically modified T cell of any of the modalities 1 to 11, wherein said at least one Cα part is encoded by less than the natural length of exon 1 of the open reading frame of the endogenous TRAC locus.

[00806] 13. The genetically modified T cell of any of modalities 1 to 12, in which the encoded TCRα chain is able to dimerize with a TCRβ chain.

[00807] 14. The genetically modified T cell of any of modalities 1 to 13, in which the encoded Cα comprises the selected sequence of any of SEQ ID NOS: 14, 15, 19, or 24, or a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS: 14, 15, 19, or 24, or a partial sequence thereof.

[00808] 15. The genetically modified T cell of any of the 6-14 modalities, in which the other part of Cα is encoded by a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of the same or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% , 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof, or a partial sequence of it.

[00809] 16. The genetically modified T cell of modality 6 to 15, in which the other part of Cα is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long.

[00810] 17. The genetically modified T cell of modality 14 or modality 15, in which the other part of Cα is encoded by a nucleotide sequence comprising less than four exons, less than three exons, less than two exons , an exon, or less than a full exon of the open reading frame of the TRAC locus.

[00811] 18. The genetically modified T cell of any of modalities 6-17, in which the other part of Cα is encoded by an exon 1 part of the TRAC locus, in which the exon 1 part is less than than the natural size of exon 1 of the open reading frame of the TRAC locus.

[00812] 19. The genetically modified T cell of any of modalities 6 to 18 in which the other part of Cα is encoded by an exon 1 part of the TRAC locus, in which the exon 1 part comprises 5 ' exon part 1.

[00813] 20. The genetically modified T cell of any of modalities 6 to 19, wherein the other part of Cα comprises a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 142 , or a partial sequence of it.

[00814] 21. The genetically modified T cell of any of modalities 6 to 20, wherein the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one substitution, deletion, or insertion of one or more amino acids in comparison to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more bridges of non-native disulfides between the alpha chain and the beta chain.

[00815] 22. The genetically modified T cell of any of modalities 1 to 21, in which the modified T cell also comprises a genetic disruption in a TRBC locus.

[00816] 23. The genetically modified T cell of any of modalities 1 to 22, in which the modified T cell also comprises a genetic disruption in a TRBC1 locus and / or a TRBC2 locus.

[00817] 24. The genetically modified T cell of any of modalities 1 to 23, wherein the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one substitution, deletion, or insertion of one or more amino acids in comparison to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more bridges of non-native disulfides between the alpha chain and the beta chain.

[00818] 25. A genetically modified T cell, comprising a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus comprising a nucleic acid encoding a recombinant TCR or part thereof, the said recombinant TCR or part thereof comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a constant domain, and (ii) an alpha TCR chain (TCRα) comprising a variable alpha domain (Vα) and a constant domain, where the nucleic acid sequence comprises (a) a transgene sequence encoding the TCRα and the Vβ domain, and (b) and an open reading frame for the locus Endogenous TRBC or a partial sequence thereof, in which the open reading frame encodes at least a part of the recombinant TCR Cβ domain.

[00819] 26. The genetically modified T cell of modality 25, in which the transgene sequence is integrated by means of homology-directed repair (HDR).

[00820] 27. The genetically modified T cell of modality 25 or modality 26, wherein the TRBC locus is a TRBC1 locus and / or a TRBC2 locus.

[00821] 28. The genetically modified T cell of modality 25 or modality 26, in which the modified TRBC locus comprises a fusion in structure of (i) a transgene sequence and (ii) and an open reading structure or a partial sequence of that of the endogenous TRBC locus.

[00822] 29. The genetically modified T cell of any one of embodiments 25 to 28, wherein the transgene sequence does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron.

[00823] 30. The genetically modified T cell of any of the modalities 25 to 29, wherein the open reading frame or a partial sequence thereof comprises the 3 'RTU of the endogenous TRBC locus.

[00824] 31. The genetically modified T cell of any of the modalities 25 to 30, in which a part of the Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of the Cβ is encoded by the transgene sequence, in which the other part of Cβ is smaller than the natural size of a native Cβ.

[00825] 32. The genetically modified T cell of any of the modalities 25 to 31, wherein the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus.

[00826] 33. The genetically modified T cell of any of the modalities 25 to 32, in which the transgene sequence is in structure with one or more exons of the open reading frame or partial sequence of the same of the endogenous TRBC locus.

[00827] 34. The genetically modified T cell of any of the modalities 25 to 33, in which the transgene sequence is integrated downstream of the nucleotide plus 5 'of exon 1 and upstream of the nucleotide plus 3' of exon 1 of the open reading structure of the endogenous TRBC locus.

[00828] 35. The genetically modified T cell of any of the modalities 25 to 34, wherein said at least a part of Cβ is encoded by at least exons 2-4 of the open reading frame of the endogenous TRBC locus.

[00829] 36. The genetically modified T cell of any of the modalities 25 to 35, in which said at least one Cβ part is encoded by at least one part of exon 1 and exons 2 to 4 of the open reading frame of the endogenous TRBC locus.

[00830] 37. The genetically modified T cell of any of the modalities 25 to 36, wherein said at least one Cβ part is encoded by less than the natural length of exon 1 of the open reading frame of the endogenous TRBC locus.

[00831] 38. The genetically modified T cell of any of the modalities 25 to 37, in which the encoded TCRβ chain is able to dimerize with the TCRα chain.

[00832] 39. The genetically modified T cell of any of modalities 25 to 38, wherein the encoded Cβ comprises the selected sequence of any of SEQ ID NO: 16, 17, 21, or 25, or a sequence showing at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 16, 17, 21, or 25, or a partial sequence thereof.

[00833] 40. The genetically modified T cell of any of the modalities 31-39, in which the other part of Cα is encoded by:

[00834] a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof, or a partial sequence thereof; or

[00835] a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof, or a partial sequence thereof.

[00836] 41. The genetically modified T cell of modality 31 to 40, in which the other part of Cα is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long.

[00837] 42. Genetically modified T cell of modality 35 or modality 36, in which the other part of Cα is encoded by a nucleotide sequence comprising less than four exons, less than three exons, less than two exons , an exon, or less than an exon complete the open reading frame of a TRBC locus.

[00838] 43. The genetically modified T cell of any of modalities 31 to 42, in which the other part of Cα is encoded by an exon 1 part of a TRBC locus, in which exon 1 part is less than the natural size of exon 1 of the open reading frame of the TRBC locus.

[00839] 44. The genetically modified T cell of any of modalities 31 to 43 in which the other part of Cα is encoded by an exon 1 part of a TRBC locus, in which the exon 1 part comprises a 5 'part of exon 1.

[00840] 45. The genetically modified T cell of any of modalities 25 to 44, wherein the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one substitution, deletion, or insertion of one or more amino acids in comparison to a native Cβ region and / or a native Cα region, optionally those referred to one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain.

[00841] 46. The genetically modified T cell of any of the modalities 25 to 45, in which the modified T cell also comprises a genetic disruption in a TRAC locus.

[00842] 47. The genetically modified T cell of any one of modalities 1 to 46, in which the transgene sequence comprises one or more multicistronic elements.

[00843] 48. The genetically modified T cell of modality 47, in which the multicistronic elements are positioned between the nucleic acid sequence that encodes TCRα or a part of it and the nucleic acid sequence that encodes TCRβ or a part the same.

[00844] 49. The genetically modified T cell of modality 47 or modality 48, wherein said one or more multi-cistronic elements are upstream of the nucleic acid sequence encoding the TCR or a part of the TCR or the molecule nucleic acid encoding a TCR.

[00845] 50. The genetically modified T cell of any of the modalities 47 to 49, in which the multicistronic element is or comprises a ribosome skip sequence, optionally T2A, P2A, E2A, or F2A.

[00846] 51. The genetically modified T cell of any of modalities 1 to 50, also comprising one or more heterologous control or regulatory elements.

[00847] 52. The genetically modified T cell of any of modalities 1 to 51, wherein the transgene sequence comprises one or more heterologous or regulatory control elements operably linked to control TCR expression when expressed from an introduced cell with the genetically modified T cell.

[00848] 53. The genetically modified T cell of modality 51 or modality 52, wherein said one or more heterologous control or regulatory elements comprises a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, a splice acceptor sequence and / or a splice donor sequence.

[00849] 54. The genetically modified T cell of any of modalities 51 to 53, in which the heterologous or control regulatory element comprises a heterologous promoter.

[00850] 55. Genetically modified T cell of modality 54, in which the heterologous promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and / or a tissue specific promoter.

[00851] 56. The genetically modified T cell of modality 54 or modality 55, wherein the heterologous promoter is or comprises a human elongation factor 1 alpha promoter or an MND promoter or a variant thereof.

[00852] 57. The genetically modified T cell of any of modalities 1 to 56, wherein the Cα and / or Cβ of the recombinant TCR comprises one or more non-native cysteines.

[00853] 58. The genetically modified T cell of any of modalities 1 to 57, in which the recombinant TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition.

[00854] 59. The genetically modified T cell of any of modalities 1 to 58, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer.

[00855] 60. The genetically modified T cell of any of modalities 1 to 59, in which the antigen is a tumor antigen or a pathogenic antigen.

[00856] 61. The genetically modified T cell of modality 60, in which the pathogenic antigen is a bacterial antigen or viral antigen.

[00857] 62. The genetically modified T cell of modality 61, in which the antigen is a viral antigen, optionally a viral antigen of hepatitis A, hepatitis B, hepatitis C virus (HCV), human papilloma virus (HPV) , hepatitis viral infections, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T cell leukemia virus 1 (HTLV-1), human T cell leukemia virus 2 (HTLV) -2), or cytomegalovirus (CMV).

[00858] 63. The genetically modified T cell of modality 61 or modality 62, in which the viral antigen is an antigen of an HPV selected from HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35.

[00859] 64. The genetically modified T cell of modality 61 or modality 62, in which the viral antigen is an HPV-16 antigen which is an HPV-16 E6 or HPV-16 E7 antigen.

[00860] 65. The genetically modified T cell of modality 61 or modality 62, in which the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA) -1, EBNA-2, EB-NA-3A , EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA.

[00861] 66. The genetically modified T cell of modality 61 or modality 62, wherein the viral antigen is an HTLV antigen which is TAX.

[00862] 67. Genetically modified T cell of modality 61 or modality 62, in which the viral antigen is an HBV antigen that is a core antigen of hepatitis B or an envelope antigen of hepatitis B.

[00863] 68. Genetically modified T cell of modality 61 or modality 62, in which the antigen is a tumor antigen.

[00864] 69. The genetically modified T cell of modality 59 or 60, in which the antigen is selected from the antigen associated with the glioma, β-human chorionic gonadotropin, alpha fetoprotein (AFP), AFP reactive to lectin, thyroglobulin, RAGE-1 , MN-CA IX, human telomerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, Melanin-A / MART-1, WT-1, S-100, MBP, CD63, MUC1 (e.g., MUC1-8), p53, Ras, cyclin B1, HER-2 / neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4 , MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE -C1, BAGE, GAGE-1, GAGE-

2, p5, tyrosinase (for example, tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β-catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, p78 IFN-inducible, melanotransferrin (p97), Uroplaquin II, prostate-specific antigen (PSA) , human kallikrein (huK2), prostate specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH -IGK, MYL-RAR, Caspase 8, FRa, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22 , ROR1, CD33 / IL3Ra, c- Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF) -I, IGF-II, IGF-I receptor and mesothelin.

[00865] 70. The genetically modified T cell of any of modalities 1 to 69, wherein the T cell is a primary T cell derived from an individual, optionally in which the individual is a human.

[00866] 71. The genetically modified T cell of any of modalities 1 to 70, wherein the T cell is a CD8 + T cell or subtypes thereof.

[00867] 72. The genetically modified cell of any of modalities 1 to 71, wherein the T cell is a CD4 + T cell or subtypes thereof.

[00868] 73. The genetically modified cell of any of the modalities 1 to 72, in which the T cell is derived from a multipotent or pluripotent cell, which optionally is an iPSC.

[00869] 74. A composition comprising a plurality of genetically modified T cells of any of modalities 1 to 73.

[00870] 75. The composition of modality 74, which comprises

las CD4 + and / or CD8 +.

[00871] 76. The composition of modality 74 or modality 75, wherein the composition comprises CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is or about 1: 3 to 3: 1, optionally 1: 1.

[00872] 77. The composition of any of the modalities 74 to 76, in which:

[00873] at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a T cell receptor beta constant region (TRBC) gene; and / or

[00874] at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the cells modified in the composition do not express or do not express detectable levels of an endogenous TRAC or TRBC gene product.

[00875] 78. Composition of any of the modalities 74 to 77, in which at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant TCR and / or exhibit antigen binding.

[00876] 79. A polynucleotide, comprising:

[00877] (a) a nucleic acid sequence encoding a part of a recombinant T cell receptor (TCR), said nucleic acid sequence encoding (i) a beta T cell receptor (TCRβ) chain that comprises a beta variable domain (Vβ) and a beta constant domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is smaller than a native, full-size TCRα chain, and

[00878] (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homologous arms

include a sequence homologous to one or more regions of an open reading frame of a TRAC locus.

[00879] 80. The polynucleotide of modality 79, in which the TCRα chain comprises a constant alpha region (Cα), in which at least a part of said Cα is encoded by the open reading structure of the endogenous TRAC locus or a sequence partial of it when the TCR or antigen-binding fragment of it is expressed from a cell introduced with the polynucleotide.

[00880] 81. The polynucleotide of modality 79 or 80, wherein the nucleic acid sequence of (a) and said one or more of the homology arms together comprise a nucleotide sequence that encodes the Cα that is smaller than that the natural size of a native Cα, in which at least a part of the Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed at from a cell introduced with the polynucleotide.

[00881] 82. The polynucleotide of any of the modalities 79 to 81, wherein the polynucleotide is comprised in a viral vector.

[00882] 83. The polynucleotide of any one of embodiments 79 to 82, wherein the nucleic acid sequence encoding the TCRβ chain is upstream of the nucleic acid sequence encoding a part of the TCRα chain.

[00883] 84. The polynucleotide of any one of embodiments 79 to 83, wherein the nucleic acid sequence of (a) does not comprise an intron.

[00884] 85. The polynucleotide of any one of modalities 79 to 84, wherein the nucleic acid sequence of (a) is a sequence that is exogenous or heterologous to an open reading frame of an endogenous genomic TRAC locus of a cell T, optionally a human T cell.

[00885] 86. The polynucleotide of any one of modalities 79 to 85, in which the nucleic acid sequence of (a) is in structure with one or more exons or a partial sequence thereof of the open reading structure of the locus TRAC comprised in said one or more homology arms.

[00886] 87. The polynucleotide of any of the modalities 79 to 86, wherein said one or more exons or a partial sequence thereof of the open reading frame comprises the sequence within exon 1 of the open reading frame of the TRAC locus.

[00887] 88. The polynucleotide of any of the modalities 79 to 87, in which the TCRα chain is able to dimerize with a TCRβ chain, when produced from a cell introduced with the polynucleotide.

[00888] 89. The polynucleotide of any of the modalities 79 to 88, wherein the part of the TCRα chain comprises a variable alpha domain (Vα).

[00889] 90. The polynucleotide of any of the modalities 79 to 89, in which a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of the other Cα part is encoded by the nucleic acid sequence of (a), wherein said other part of Cα is smaller than the natural size of a native Cα.

[00890] 91. The polynucleotide of modality 90, in which the other part of Cα is encoded by a nucleotide sequence starting from residue 3 and until residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of the same or a sequence that displays at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof, or a partial sequence thereof.

[00891] 92. Polynucleotide of modality 91, in which the other part of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long.

[00892] 93. Polynucleotide of modality 91 or 92, in which the other part of Cα is encoded by a nucleotide sequence comprising less than four exons, less than three exons, less than two exons, one exon, or less than a full exon of the open reading frame of the TRAC locus.

[00893] 94. The polynucleotide of any of the modalities 91 to 93, in which the other part of Cα is encoded by an exon 1 part of the TRAC locus, in which exon 1 part is smaller than the natural size of exon 1 of the open reading frame of the TRAC locus.

[00894] 95. The polynucleotide of any of the modalities 90 to 94, wherein the other part of Cα is encoded by an exon 1 part of the TRAC locus, wherein the exon 1 part comprises a 5 'part of exon 1 .

[00895] 96. The polynucleotide of any of the 79 to 95 modalities, wherein the other part of the Cα comprises a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 142, or a partial sequence of it.

[00896] 97. The polynucleotide of any of the modalities 79 to 96, wherein the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids compared to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more more non-native disulfide bridges between the alpha chain and the beta chain.

[00897] 98. The polynucleotide of any one of embodiments 79 to 97, wherein said one or more homology arms comprise a 5 'homology arm and / or a 3' homology arm.

[00898] 99. The 98-mode polynucleotide, wherein the 5 'homology arm and the 3' homology arm comprise nucleic acid sequences homologous to the nucleic acid sequences surrounding a target site, where the target site is within the TRAC locus.

[00899] 100. Polynucleotide of modality 99, wherein the target site is within exon 1 of the TRAC locus.

[00900] 101. The polynucleotide of any of the modalities 98 to 100, wherein the 5 'homology arm comprises:

[00901] a) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides contiguous to a sequence that exhibits at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the sequence men - listed in SEQ ID NO: 124;

[00902] b) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence mentioned in SEQ ID NO: 124; or

[00903] c) the sequence mentioned in SEQ ID NO: 124.

102. The polynucleotide of any of the modalities 98 to 101, wherein the homology arm 3 'comprises:

[00905] a) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides contiguous to a sequence that exhibits at least 85%,

86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the sequence mentioned in SEQ ID NO: 125;

[00906] b) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence mentioned in SEQ ID NO: 125; or

[00907] c) the sequence mentioned in SEQ ID NO: 125.

[00908] 103. A polynucleotide, comprising:

[00909] (a) a nucleic acid sequence encoding a part of a recombinant T cell receptor (TCR), said nucleic acid sequence encoding (i) an alpha T cell receptor (TCRα) chain comprising a variable alpha domain (Vα) and a constant alpha domain (Cα); and (ii) a part of a T-cell receptor (TCRβ) beta chain, where the part of the TCRβ chain is smaller than a full-size native TCRβ chain, and

[00910] (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homology arms comprise a sequence homologous to one or more regions of an open reading frame of a TRBC locus .

[00911] 104. The polynucleotide of modality 103, in which the TCRβ chain comprises a beta constant (Cβ), in which at least a part of said Cβ is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence the same when the TCR or antigen-binding fragment of it is expressed from a cell introduced with the polynucleotide.

[00912] 105. The polynucleotide of modality 103 or 104, wherein the nucleic acid sequence of (a) and said one or more homology arms together comprise a nucleotide sequence that encodes a Cβ that is smaller than that the natural size of a native Cβ, in which at least part of the Cβ is encoded

each by the open reading frame of the endogenous TRBC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide.

[00913] 106. The polynucleotide of any of the modalities 103 to 105, wherein the polynucleotide is comprised in a viral vector.

[00914] 107. The polynucleotide of any of the modalities 103 to 106, wherein the TRBC locus is one or more of TRBC1 or TRBC2.

[00915] 108. The polynucleotide of any of the modalities 103 to 107, wherein the nucleic acid sequence encoding the TCRα chain is upstream of the nucleic acid sequence encoding the part of the TCRβ chain.

[00916] 109. The polynucleotide of any of the modalities 103 to 108, wherein the nucleic acid sequence of (a) does not comprise an intron.

[00917] 110. The polynucleotide of any of the modalities 103 to 109, wherein the nucleic acid sequence of (a) is a sequence that is exogenous or heterologous to an open reading frame of an endogenous genomic TRBC locus of a T cell, optionally a human T cell.

[00918] 111. The polynucleotide of any of the modalities 103 to 110, in which the nucleic acid sequence of (a) is in structure with one or more exons or a partial sequence thereof of the open reading structure of the locus TRAC comprised in said one or more homology arms.

[00919] 112. The polynucleotide of any of the modalities 103 to 111, wherein said one or more exons or a partial sequence thereof of the open reading frame is or comprises a sequence within exon 1 of the open reading frame of the locus

TRBC.

[00920] 113. The polynucleotide of any of the modalities 103 to 112, in which the TCRβ chain is able to dimerize with a TCRα chain, when produced from a cell introduced with the polynucleotide.

[00921] 114. The polynucleotide of any of the modalities 103 to 113, wherein the part of the TCRβ chain comprises a variable beta domain (Vβ).

[00922] 115. The polynucleotide of any of the modalities 103 to 114, in which a part of the Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of the Cβ is encoded by the nucleic acid sequence of (a), in which said other part of Cβ is smaller than the natural size of a native Cβ

[00923] 116. Polynucleotide of modality 115, in which the other part of Cβ is encoded by:

[00924] a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof, or a partial sequence thereof; or

[00925] a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof, or a partial sequence thereof.

[00926] 117. Polynucleotide of modality 115 or 116, in which the other part of Cβ is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200 , less than 150, base pairs long.

[00927] 118. The polynucleotide of any of the modalities 115 to 117, in which the other part of Cβ is encoded by a sequence of nucleotides that encodes less than four exons, less than three exons, less than two exons, an exon, or less than a full exon of the open reading frame of the TRBC locus.

[00928] 119. The polynucleotide of any of the modalities 115 to 118, in which the other part of the Cβ is encoded by an exon 1 part of a TRBC locus, in which the exon 1 part is smaller than the natural size exon 1 of the open reading frame of the TRBC locus.

[00929] 120. The polynucleotide of modalities 115 to 119, wherein the other part of the TCRα constant domain is encoded by an exon 1 part of the TRAC locus, wherein the exon 1 part comprises a 5 'part of exon 1.

[00930] 121. The polynucleotide of any of the modalities 115 to 120, wherein the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally a substitution, deletion , or insertion of one or more amino acids compared to a native Cβ region and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more disulfide bridges non-native between the alpha chain and the beta chain.

[00931] 122. The polynucleotide of any of the modalities

115 to 121, wherein said one or more homology arms comprise a 5 'homology arm and / or a 3' homology arm.

[00932] 123. The polynucleotide of modality 122, wherein the 5 'homology arm and the 3' homology arm comprise nucleic acid sequences homologous to the nucleic acid sequences surrounding a target site, where the target site is within the open reading structure of the TRBC locus.

[00933] 124. The polynucleotide of modality 123, wherein the target site is within exon 1 of the open reading frame of the TRBC locus.

[00934] 125. The polynucleotide of any of the modalities 98 to 100 and 122 to 124, wherein the homology arm 5 'and the homology arm 3' independently are at least or at least about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides, or less than or less than about 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, or 2000 nucleotides.

126. The polynucleotide of modality 125, wherein the 5 'homology arm and the 3' homology arm independently of or from about 100 to 1000 nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides , 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides, 600 to 750 nucleotides or 750 to 1000 nucleotides in length.

127. The polynucleotide of modality 125 or modality 126, wherein the homology arm 5 'and the homology arm 3' independently

they are, are about, or are less than about 600 nucleotides in length.

[00937] 128. The polynucleotide of any of the modalities 79 to 127, wherein the nucleic acid sequence of (a) comprises one or more multicistronic elements.

[00938] 129. The polynucleotide of modality 128, in which the multicistronic elements are positioned between the nucleic acid sequence that encodes TCRα or a part of it and the nucleic acid sequence that encodes TCRβ or a part of it.

[00939] 130. The polynucleotide of mode 128 or mode 129, wherein said one or more multicistronic elements are upstream of the nucleic acid sequence encoding the TCR or a part of the TCR or the nucleic acid molecule encoding a TCR .

[00940] 131. The polynucleotide of either 129 or 130, wherein the multicistronic element is or comprises a ribosome jump sequence, optionally T2A, P2A, E2A, or F2A.

[00941] 132. The polynucleotide of any of the modalities 79 to 131, also comprising one or more heterologous control or regulatory elements.

[00942] 133. The polynucleotide of any one of modalities 79 to 132, wherein the nucleic acid sequence of (a) comprises one or more heterologous or regulatory control elements operably linked to control TCR expression when expressed as a cell introduced with the polynucleotide.

[00943] 134. Polynucleotide of modality 132 or 133 in which said one or more heterologous control or regulatory elements comprises the promoter, an enhancer, an intron, a polyadenylation signal, a consensus sequence, an acceptor sequence and / or a splice donor sequence.

[00944] 135. The polynucleotide of any of the modalities 132 to 134, wherein the heterologous or control regulatory element comprises a heterologous promoter.

[00945] 136. Polynucleotide of modality 135, wherein the heterologous promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and / or a tissue-specific promoter.

[00946] 137. Polynucleotide of modality 135 or modality 136, wherein the heterologous promoter is or comprises a human elongation factor 1 alpha promoter or an MND promoter or a variant thereof.

[00947] 138. The polynucleotide of any of the modalities 82 to 102 or 106 to 137, wherein the viral vector is an AAV vector.

[00948] 139. Polynucleotide of modality 138, in which the vector AAV is selected from the vector AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.

[00949] 140. Polynucleotide of modality 139, wherein the AAV vector is an AAV2 or AAV6 vector.

[00950] 141. The polynucleotide of any of the modalities 82 to 102 or 106 to 137, wherein the viral vector is a retroviral vector.

[00951] 142. The polynucleotide of any of the modalities 82 to 102 or 106 to 137, wherein the viral vector is the lentiviral vector.

[00952] 143. The polynucleotide of any of the modalities 79 to 81, 84 to 105, or 107 to 137, wherein the polynucleotide is a linear polynucleotide.

[00953] 144. Polynucleotide of modality 143, where in the linear polynucleotide is a double-stranded polynucleotide.

[00954] 145. Polynucleotide of modality 144, where in the linear polynucleotide is a single-stranded polynucleotide.

[00955] 146. The polynucleotide of any of the modalities 79 to 145, wherein the polynucleotide comprises the structure: [5 'homology arm] - [nucleic acid sequence of (a)] - [homology arm 3 '].

[00956] 147. The polynucleotide of any of the modalities 128 to 146, in which the polynucleotide comprises the structure: [5 'homology arm] - [multicistronic element] - [nucleic acid sequence of (a)] - [3 'homology arm].

[00957] 148. The polynucleotide of any of the modalities 134 to 147, wherein the polynucleotide comprises the structure: [5 'homology arm] - [promoter] - [nucleic acid sequence of (a)] - [arm of 3 'homology].

[00958] 149. The polynucleotide of any of the modalities 79 to 148, in which the recombinant TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition.

[00959] 150. Polynucleotide modality 149, wherein the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer.

[00960] 151. Polynucleotide of modality 149, in which the antigen is a tumor antigen or a pathogenic antigen.

[00961] 152. Polynucleotide of modality 151, in which the pathogenic antigen is a bacterial antigen or viral antigen.

[00962] 153. Polynucleotide of modality 152, wherein the antigen is a viral antigen, optionally a hepatitis A viral antigen, hepatitis B, hepatitis C virus (HCV), human papilloma virus (HPV), viral infections hepatitis, Epstein-Barr virus (EBV), human herpes virus 8 (HHV-8), human T cell leukemia virus 1 (HTLV-1), human T cell leukemia virus 2 (HTLV- 2), or cytomegalovirus (CMV).

[00963] 154. Polynucleotide of modality 152 or 153 in which the antigen is an antigen from an HPV selected from HPV-16, HPV-18, HPV-31, HPV-33 and HPV-35.

[00964] 155. Polynucleotide of modality 152 or 153, wherein the antigen is an HPV-16 antigen which is an HPV-16 E6 or HPV-16 E7 antigen.

[00965] 156 Polynucleotide of modality 152 or 153, in which the viral antigen is an EBV antigen selected from Epstein-Barr nuclear antigen (EBNA) -1, EBNA-2, EBNA-3A, EBNA-3B, EBNA-3C, EBNA leader protein (EBNA-LP), latent membrane proteins LMP-1, LMP-2A and LMP-2B, EBV-EA, EBV-MA and EBV-VCA.

[00966] 157. Polynucleotide of modality 152 or 153, wherein the viral antigen is an HTLV antigen which is TAX.

[00967] 158. Polynucleotide of modality 152 or 153, wherein the viral antigen is an HBV antigen that is a core antigen of hepatitis B or an envelope antigen of hepatitis B.

[00968] 159. Polynucleotide of modality 151, wherein the antigen is a tumor antigen.

[00969] 160. Polynucleotide of modality 159, in which the antigen is selected from antigen associated with glioma, β-human chorionic gonadotropin, alpha fetoprotein (AFP), AFP reactive to lectin, thyroglobulin, RAGE- 1, MN-CA IX, human telerase reverse transcriptase, RU1, RU2 (AS), intestinal carboxyl esterase, mut hsp70-2, M-CSF, Melanin-A / MART-1, WT-1, S-100 , MBP, CD63, MUC1 (eg MUC1-8), p53, Ras, cyclin B1, HER-2 / neu, carcinoembryonic antigen (CEA), gp100, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10, MAGE-A11, MAGE-A11, MAGE-B1, MAGE-B2, MAGE-B3, MAGE- B4, MAGE-C1, BAGE, GAGE-1, GAGE-2, p5, tyrosinase (for example, tyrosinase-related protein 1 (TRP-1) or tyrosinase-related protein 2 (TRP-2)), β- catenin, NY-ESO-1, LAGE-1a, PP1, MDM2, MDM4, EGVFvIII, Tax, SSX2, telomerase, TARP, pp65, CDK4, vimentin, S100, eIF-4A1, p78 IFN-inducible, melanotrans-ferrine (p97 ), Uroplaquina II, antí prostate specific genus (PSA), human kallikrein (huK2), prostate specific membrane antigen (PSM), and prostatic acid phosphatase (PAP), neutrophil elastase, ephrin B2, BA-46, Bcr-abl, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Caspase 8, FRa, CD24, CD44, CD133, CD 166, epCAM, CA-125, HE4, Oval, estrogen receptor, progesterone receptor, uPA, PAI-1, CD19, CD20, CD22, ROR1, CD33 / IL3Ra, c-Met, PSMA, Glycolipid F77, GD-2, insulin growth factor (IGF) -I, IGF-II, IGF-I re - ceptor and mesothelin.

[00970] 161. A method of producing a genetically modified T cell comprising a modified TRAC locus, which comprises introducing the polynucleotide of any of the 79 to 102 or 125 to 160 modalities into a T cell comprising a genetic disruption at a TRAC locus.

[00971] 162. A method of producing a genetically modified T cell that comprises a modified alpha T cell receptor (TRAC) constant locus, the method comprising:

[00972] (a) introducing, in a T cell, one or more agents capable of inducing a genetic disruption at a target site within an endogenous T cell locus TRAC; and

[00973] (b) introducing into the T cell a polyucleotide comprising the transgene encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), wherein the part is smaller than a native, full-size TCRα chain, and in which the transgene is targeted for integration into the endogenous TRAC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRAC locus.

[00974] 163. A method of producing a genetically modified T cell that comprises a modified alpha T cell receptor (TRAC) constant locus, the method of introducing a polynucleotide into a T cell comprising the transgene encoding a T cell receptor beta chain (TCRβ) and part of a T cell receptor alpha chain (TCRα), said cell having a genetic disruption within the endogenous T cell TRAC locus, in that the transgene is targeted for integration within the endogenous TRAC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRAC locus.

[00975] 164. Method 163, in which genetic disruption was induced by one or more agents capable of inducing a genetic disruption of one or more target sites within the endocrine TRAC locus.

[00976] 165. The method of any of the modalities 161 to 164, wherein the polynucleotide is of any of the modalities 79 to 102 or 125-160.

[00977] 166. The method of any of the modalities 161 to 165, wherein the modified TRAC locus comprises a nucleic acid sequence encoding a recombinant TCR or part thereof, wherein the nucleic acid sequence comprises a spherical fusion structure of (i) a transgene sequence and (ii) and an open reading frame or a partial sequence of the same of the endogenous TRAC locus.

[00978] 167. The method of any of the modalities 161 to 166, wherein the transgene sequence does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron.

[00979] 168. The method of any of the modalities 161 to 167 wherein the open reading frame or a partial sequence thereof comprises the 3 'RTU of the endogenous TRAC locus.

[00980] 169. The method of any of the modalities 161 to 168, in which part of the Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of the Cα is encoded by the sequence transgene, wherein said other part of Cα is smaller than the natural size of a native Cα.

[00981] 170. The method of any of the modalities 161 to 169, wherein the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus.

[00982] 171. The method of any of the modalities 161 to 170, in which the transgene sequence is in structure with one or more exons of the open reading frame or partial sequence thereof of the endogenous TRAC locus.

[00983] 172. The method of any of the modalities 161 to 171, wherein the transgene sequence is integrated downstream of the nucleotide plus 5 'of exon 1 and upstream of the nucleotide plus 3' of exon 1 of the structure. open reading of the endogenous TRAC locus.

[00984] 173. The method of any of the modalities 161 to 172, wherein said at least a part of Cα is encoded by at least exons 2-4 of the open reading structure of the endogenous TRAC locus.

[00985] 174. The method of any of the modalities 161 to 173, wherein the encoded Cα comprises the sequence selected from any of SEQ ID NOS: 14, 15, 19, or 24, or a sequence that exhibits at least 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity

with any of SEQ ID NOS: 14, 15, 19, or 24, or a partial sequence of them.

[00986] 175. The method of any of the modalities 169 to 174, wherein the other part of Cα comprises a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87 %, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 142, or a partial sequence of it.

[00987] 176. The method of any of the modalities 161 to 175, wherein the modified T cell also comprises inducing a genetic disruption in a TRBC locus.

[00988] 177. The method of any of the modalities 161 to 174, wherein the modified T cell comprises a genetic disruption at a TRBC1 locus and / or a TRBC2 locus.

[00989] 178. A method of producing a genetically modified T cell comprising a modified TRBC locus, which comprises introducing the polynucleotide of any of the modalities 103 to 160 into a T cell comprising a genetic disruption in a TRBC locus.

[00990] 179. A method of producing a genetically modified T cell comprising a modified T cell receptor (TRBC) beta constant locus, the method comprising:

[00991] (a) introducing, in a T cell, one or more agents capable of inducing a genetic rupture at a target site within an endogenous TRBC locus of the T cell; and

[00992] (b) introducing into the T cell a polyucleotide comprising the transgene encoding a T cell receptor alpha chain (TCRα) and a part of a T cell receptor beta chain (TCRβ), wherein the part is smaller than a native, full-size TCRβ chain, and in which the transgene is targeted for integration into an endogenous TRBC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRBC locus .

[00993] 180. A method of producing a genetically modified T cell comprising a modified T cell receptor beta (TRBC) constant locus, the method comprising introducing, into a T cell, a polynucleotide comprising the transgene encoding a T cell receptor alpha chain (TCRα) and part of a T cell receptor beta chain (TCRβ), that cell having a genetic disruption within an endogenous TRBC locus of T cell, in which the transgene is directed to integration within the endogenous TRBC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRBC locus.

[00994] 181. The method of modality 180, in which the genetic disruption was induced by one or more agents capable of inducing a genetic disruption of one or more target sites within the endogenous TRBC locus.

[00995] 182. The method of any of the modalities 178 to 181, wherein the TRBC locus is a TRBC1 locus and / or a TRBC2 locus.

[00996] 183. The method of any of the modalities 178 to 182, wherein the polynucleotide is of any of the modalities 95 to 149.

[00997] 184. The method of any of the modalities 178 to 183, wherein the modified TRBC locus comprises a nucleic acid sequence encoding a recombinant TCR or part thereof, wherein the nucleic acid sequence comprises a fusion in structure of (i) a transgene sequence and (ii) and an open reading frame or a partial sequence thereof of the endogenous TRBC locus.

[00998] 185. The method of any of the modalities 178 to 184, wherein the transgene sequence does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron.

186. The method of any of the modalities 178 to 185, wherein the open reading frame or a partial sequence thereof comprises the 3 'RTU of the endogenous TRBC locus.

[001000] 187. The method of any of the modalities 178 to 186, in which a part of the Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of the Cβ is encoded by the sequence of transgene, in which said other part of Cβ is smaller than the natural size of a native Cβ.

188. The method of any of the modalities 178 to 187, in which the open reading structure or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus.

189. The method of any of the modalities 178 to 188, in which the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence thereof of the endogenous TRBC locus.

[001003] 190. The method of any of the modalities 178 to 189, wherein the transgene sequence is integrated downstream of the nucleotide plus 5 'of exon 1 and upstream of the nucleotide plus 3' of exon 1 of the structure. open reading of the endogenous TRBC locus.

[001004] 191. The method of any of the modalities 178 to 190, wherein said at least a part of Cβ is encoded by at least exons 2-4 of the open reading structure of the endogenous TRBC locus.

[001005] 192. The method of any of the modalities 178 to 191, wherein the encoded Cβ comprises the sequence selected from any of SEQ ID NO: 16, 17, 21, or 25, or a sequence that exhibits at least 85 %, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 16, 17, 21, or 25, or a partial sequence thereof.

[001006] 193. The method of any of the modalities 177 to 192, wherein the modified T cell also comprises inducing a genetic disruption in a TRAC locus.

[001007] 194. The method of any of the modalities, wherein said one or more agents capable of inducing a genetic disruption comprises a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site.

[001008] 195. Method 194 method, wherein said one or more agents capable of inducing a genetic disruption comprises (a) a fusion protein comprising a DNA targeting protein and a nuclease or (b) an RNA-guided nuclease.

[001009] 196. Method method 195, wherein the RNA-guided DNA or Nuclease protein comprises a zinc finger protein (ZFP), a TAL protein, or a regularly associated short palindromic nucleic acid interspersed in cluster (CRISPR) (Cas) specific to the target site.

197. The method of any of the modalities 194 to 196, wherein said one or more agents comprise a zinc finger nucleus (ZFN), a TAL effector nuclease (TALEN), or and a combination of CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes to the target site.

[001011] 198. The method of any of the modalities 162, 164 to 177, 179, or 181 to 197, wherein each of the said one or more agents comprise a guide RNA (gRNA) having a targeting domain that is complement at least one target site.

199. Method 198 method, wherein said one or more agents are introduced as a ribonucleo-protein (RNP) complex comprising gRNA and a Cas9 protein.

[001013] 200. Method 199, in which RNP is introduced by means of electroporation, particle gun, calcium phosphate transfection, compression or cellular squeezing.

[001014] 201. Method 199 or method 200, in which RNP is introduced by means of electroporation.

[001015] 202. The method of any of the 162, 164 to 177, 179, or 181 to 197 modalities, wherein said one or more agettes is introduced as one or more polynucleotides encoding a gRNA and / or a Cas9 protein .

[001016] 203. The method of any of the 198 to 202 modalities, wherein the gRNA has a targeting domain that is complementary to a target site at a TRAC locus and comprises a selected sequence of UCUCUCAGCUGGUACACGGC (SEQ ID NO: 28) , UGGAUUAGAGUCUCUCAGC (SEQ ID NO: 29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO: 30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31), GCUGGUACACGGCAGGGA (SEQ ID NO: 32), SEA ID: 32), CAGGGAC NO: 34), GCUAGACAUGAGGUCUA (SEQ ID NO: 35), GUCAGAUUUGUUGCUCC (SEQ ID NO: 36), UCAGCUGGUACACGGCA (SEQ ID NO: 37), GCAGACAGACUUGUCAC (SEQ ID NO: 38), GGUACACGGCAGGGA: CUUCAAGAGCAACAGUGCUG (SEQ ID NO: 40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO: 41),

AAAGUCAGAUUUGUUGCUCC (SEQ ID NO: 42), ACAAAACUGUGCUAGACAUG (SEQ ID NO: 43), AAACUGUGCUAGACAUG (SEQ ID NO: 44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO: 45), GGCUGGU : 47), GGGGAAGAAGGUGUCUUC (SEQ ID NO: 48), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO: 49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO: 50), GCAGACAGACUUGUCACUGGAU (SEQ ID NO: 51), GAGGUACU (SEQ ID NO: 53), GAAUAGGCAGACAGACUUGUCA (SEQ ID NO: 54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO: 55), GUCUCUCAGCUGGUACACGG (SEQ ID NO: 56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO: 56) 58).

[001017] 204. Method 203 method, wherein the gRNA has a targeting domain comprising the sequence GAGAAU- CAAAAUCGGUGAAU (SEQ ID NO: 31).

[001018] 205. The method of any of the 198-202 modalities, wherein the gRNA has a targeting domain that is complementary to a target site in one or both of a TRBC1 and a TRBC2 gene and comprises a selected sequence of CACCCAGAUCGUCAGCGCCG (SEQ ID NO: 59), CAAACACAGCGACCUCGGGU (SEQ ID NO: 60), UGACGAGUGGACCCAGGAUA (SEQ ID NO: 61), GGCUCUCGGAGAAUGACGAG (SEQ ID NO: 62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO: SECC ID: 63) 64),

AUGACGAGUGGACCCAGGAU (SEQ ID NO: 65), AGUCCAGUUCUACGGGCUCU (SEQ ID NO: 66), CGCUGUCAAGUCCAGUUCUA (SEQ ID NO: 67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO: 68), UCAAACACAGAGGGGACGACA : 70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO: 71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO: 72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO: 73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO: 74), SEA ID: 74), GAGGU (SEQ ID NO: 76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO: 77), GGCUCAAACACAGCGACCUC (SEQ ID NO: 78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO: 79), AGGCUUCUACCCCGACCACG (SEQAGCCACACG (SEQAG NO: 80) 81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO: 82), CUUGACAGCGGAAGUGGUUG (SEQ ID NO: 83), AGAUCGUCAGCGCCGAGGCC (SEQ ID NO: 84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO: 85), UGAGGGGGGGCGGGGGGGCGGGC SEQ ID NO: 87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO: 88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO: 89), GCGGGGUUCUGCCAGAAGG (SEQ ID NO: 90), UGGCUCAAACACAGCGACCU (SEQ ID NO: 91) CUGGACUUGACAGCGGAAG (SEQ ID NO: 92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO: 93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO: 94),

GUAUCUGGAGUCAUUGAGGG (SEQ ID NO: 95), CUCGGCGCUGACGAUCU (SEQ ID NO: 96), CCUCGGCGCUGACGAUC (SEQ ID NO: 97), CCGAGAGCCCGUAGAAC (SEQ ID NO: 98), CCAGAUCGUCAGCUGAC NO (SEQ): SEQ ID: 98 : 100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 102), GUGACAGGUUUGGCCCUAUC (SEQ ID NO: 103), GACAGGUUUGGCCCUAUC (SEQ ID NO: 104), SEAGGGGACGAC (SEQ ID NO: 106), GUGGAGCUGAGCUGGUGG (SEQ ID NO: 107), GGGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 109), GCGGGCUGCUCCU 111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO: 112), GCUGCUCCUUGAGGGGCU (SEQ ID NO: 113), GGUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: AGAGGA) (SEQ ID NO: 115)

[001019] 206. The 205 method method, wherein the gRNA has a targeting domain comprising the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63).

[001020] 207. The method of modality of any of the modes 161 to 206, wherein the T cell is a CD8 + T cell or subtypes thereof.

[001021] 208. Method 207 method, wherein the T cell is a CD4 + T cell or subtypes thereof.

[001022] 209. The method of any of the modalities 161 to 206, in which the T cell is derived from a multipotent or pluripotent cell, which optionally is an iPSC.

[001023] 210. The method of any of the modalities 161 to 209, wherein the T cell comprises a T cell that is autologous to the individual.

[001024] 211. The method of any of the modalities 161 to 209, wherein the T cell comprises a T cell that is allogeneic to the individual.

[001025] 212. The method of any of the modalities 161 to 211, wherein the polynucleotide and / or the one or more polynucleotide encoding a gRNA and / or a Cas9 protein is comprised of one or more vectors, which are optionally vectors viral.

[001026] 213. The 212 method, in which the vector is an AAV vector.

[001027] 214. The method of modality 213, in which the vector AAV is selected from the vector AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8.

[001028] 215. The method of modality 214, in which the AAV vector is an AAV2 or AAV6 vector.

[001029] 216. Method 212, wherein the viral vector is a retroviral vector.

[001030] 217. The method of modality 216, wherein the viral vector is the lentiviral vector.

[001031] 218. The method of any of the modalities 161 to 211, wherein the polynucleotide is a linear polynucleotide.

[001032] 219. The method of modality 218, where in the linear polynucleotide is a double-stranded polynucleotide.

[001033] 220. The method of modality 219, where in the linear polynucleotide is a single-stranded polynucleotide.

[001034] 221. The method of any of the modalities 161 to 220, wherein the introduction of said one or more agents capable of inducing a genetic disruption and an introduction of the standard polynucleotide are carried out simultaneously or sequentially, in any order.

[001035] 222. The method of any of the modalities 161 to 221, in which the introduction of the standard polynucleotide is carried out after the introduction of said one or more agents capable of inducing a genetic disruption.

[001036] 223. Method method 222, wherein the standard polynucleotide is introduced immediately after, or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of one or more 23 capable agents to induce a genetic disruption.

[001037] 224. A modified T cell or a plurality of modified T cells generated using the method of any of the 161 to 2 modes.

[001038] 225. A composition, comprising the modified T cell or a plurality of modified cells of modality 224.

[001039] 226. The 225 mode composition, comprising CD4 + and / or CD8 + T cells.

[001040] 227. The composition of mode 225 or mode 226, wherein the composition comprises CD4 + and CD8 + T cells and the ratio of CD4 + to CD8 + T cells is from or about 1: 3 to 3: 1, opci - onally 1: 1.

[001041] 228. The composition of any of the modalities 225 to 227, in which:

[001042] at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%,

95%, 96%, 97%, 98%, or 99% of the modified cells in the composition comprise a genetic break in or an endogenous T cell receptor (TRC) alpha constant region gene and / or a constant region gene beta T cell receptor (TRBC); and / or

[001043] at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express or do not express detectable levels of an endogenous TRAC or TRBC gene product.

[001044] 229. The composition of any of the modalities 225 to 228, in which at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% , 80%, or 90% of the cells in the composition express the recombinant TCR and / or exhibit antigen binding.

[001045] 230. A method of treatment which comprises administering the modified cell, a plurality of modified cells or composition of any of the modalities 224 to 229 to an individual.

[001046] 231. Use of the modified cell, a plurality of modified cells or composition of any of the modalities 224-229 for the treatment of a disease or disorder.

[001047] 232. Use of the modified cell, a plurality of modified cells or composition of any of the modalities 224 to 229 in the manufacture of a drug for the treatment of a disease or disorder.

[001048] 233. The modified cell, a plurality of modified cells or composition of any of the modalities 224 to 229 for use in the treatment of a disease or disorder.

[001049] 234. An article of manufacture comprising:

[001050] the polynucleotide of any of the modalities 79 to 102 or 116 to 149, and

[001051] one or more agents capable of inducing a genetic disruption

at a target site within a TRAC locus.

[001052] 235. An article of manufacture comprising:

[001053] a polynucleotide comprising (a) a nucleic acid sequence encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), where the part is smaller than a natural-sized native TCRα chain and (b) one or more homology arms linked to the nucleic acid sequence wherein said one or more homology arms (s) comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus, referred to as the open reading frame encoding a TCRα chain; and

[001054] one or more agents capable of inducing a genetic rupture at a target site within a TRAC locus.

[001055] 236. The article of manufacture of modality 201, in which the polynucleotide is of any of the modalities 79 to 102 or 125 to

160.

[001056] 237. An article of manufacture comprising:

[001057] the polynucleotide of any of the modalities 79 to 160, and

[001058] one or more agents capable of inducing a genetic rupture at a target site within a TRBC locus.

[001059] 238. An article of manufacture comprising:

[001060] a polynucleotide comprising (a) a nucleic acid sequence encoding a T cell receptor alpha chain (TCRα) and a part of a T cell receptor beta chain (TCRβ), where the part is smaller than a natural-sized native TCRβ chain and (b) one or more homology arms linked to the nucleic acid sequence wherein said one or more homology arms (s) comprise a sequence homologous to one or more regions of an open reading structure of a TRBC locus, the reference

the open reading frame encoding a TCRβ chain; and

[001061] one or more agents capable of inducing a genetic disruption at a target site within a TRAC locus.

[001062] 239. The article of manufacture of modality 237 or 238, in which the locus TRBC is TRBC1 and / or TRBC2.

[001063] 240. The article of manufacture of modality 237 or 238, in which the polynucleotide is of any of the modalities 103 to 160.

[001064] 241. The article of manufacture of any of modalities 234 to 241, wherein said one or more agents capable of inducing a genetic disruption comprises a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site.

[001065] 242. The article of manufacture of modality 241, wherein said one or more agents capable of inducing a genetic disruption comprises (a) a fusion protein comprising a DNA targeting protein and the nuclease or (b) an RNA-guided nuclease.

[001066] 243. The article of manufacture of modality 242, wherein the RNA-guided DNA or Nuclease protein comprises a zinc finger protein (ZFP), a TAL protein, or a nclease associated with the regular short palindromic nucleic acid - interspersed in a cluster (CRISPR) (Cas) specific to the target site.

[001067] 244. The article of manufacture of any of the modalities 241 to 243, wherein said one or more agents comprise a zinc finger nuclease (ZFN), a TAL effector nuclease (TALEN), or and a combination of CRISPR-Cas9 that specifically binds to, recognizes, or hybridizes to the target site.

[001068] 245. The article of manufacture of any of the modalities 241 to 244, in which each of the said one or more agents comprises a guide RNA (gRNA) having a targeting domain that is complementary to the hair least one target site.

[001069] 246. The article of manufacture of modality 241 to 245, wherein said one or more agents are introduced as a complex of ribonucleoprotein (RNP) comprising the gRNA and a Cas9 protein.

[001070] 247. The manufacturing article of modality 246, in which RNP is introduced by means of electroporation, particle gun, calcium phosphate transfection, compression or cellular squeezing.

[001071] 248. The article of manufacture of modality 246 or modality 247, in which RNP is introduced by means of electroporation.

[001072] 249. The article of manufacture of any of the modalities 245 to 248, wherein said one or more agettes is introduced as one or more polynucleotides encoding a gRNA and / or a Cas9 protein.

[001073] 250. The article of manufacture of any of the modalities 245 to 249, in which the gRNA has a targeting domain that is complementary to a target site in a TRAC locus and comprises a selected sequence of UCUCUCAGCUGGUACACGGC (SEQ ID NO: 28), UGGAUUUAGAGUCUCUCAGAGC (SEQ ID NO: 29), ACACGGCAGGGUCAGGGUUC (SEQ ID NO: 30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31), GCUGGUACACGGCAGGGUCA (SEQ ID NO: 32) (SEQ ID NO: 32) SEQ ID NO: 34), GCUAGACAUGAGGUCUA (SEQ ID NO: 35), GUCAGAUUUGUUGCUCC (SEQ ID NO: 36), UCAGCUGGUACACGGCA (SEQ ID NO: 37), GCAGACAGACUUGUCAC (SEQ ID NO: 38),

GGUACACGGCAGGGUCA (SEQ ID NO: 39), CUUCAAGAGCAACAGUGCUG (SEQ ID NO: 40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO: 41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO: 42), ACAAAUGUGUQACAAA : 44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO: 45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO: 46), GCUGGGGAAGAAGGUGUCUUC (SEQ ID NO: 47), GGGGAAGAAGGUGUCUUC (SEQ ID NO: 48), GUACU (SEQ ID NO: 50), GCAGACAGACUUGUCACUGGAUU (SEQ ID NO: 51), GACAGACUUGUCACUGGAUU (SEQ ID NO: 52), GUGAAUAGGCAGACAGACUUGUCA (SEQ ID NO: 53), GAAUAGGCAGACAGACUUGUCA (SEQ ID: 54) 55), GUCUCUCAGCUGGUACACGG (SEQ ID NO: 56), GGUACACGGCAGGGUCAGGGUU (SEQ ID NO: 57) and GUACACGGCAGGGUCAGGGUU (SEQ ID NO: 58).

[001074] 251. The article of manufacture of modality 250, in which the gRNA has a targeting domain that comprises the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31).

[001075] 252. The article of manufacture of any of the modalities 245 to 251, in which the gRNA has a targeting domain that is complementary to a target site in one or both of a TRBC1 and a TRBC2 gene and comprises a selected sequence from CACCCAGAUCGUCAGCGCCG (SEQ ID NO: 59), CAAACACAGCGACCUCGGGU (SEQ ID NO: 60), UGACGAGUGGACCCAGGAUA (SEQ ID NO: 61),

GGCUCUCGGAGAAUGACGAG (SEQ ID NO: 62), GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63), GAAAAACGUGUUCCCACCCG (SEQ ID NO: 64), AUGACGAGUGGACCCAGGAU (SEQ ID NO: 65), AGUCCGUCU : 67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO: 68), UCAAACACAGCGACCUCGGG (SEQ ID NO: 69), CGUAGAACUGGACUUGACAG (SEQ ID NO: 70), AGGCCUCGGCGCUGACGAUC (SEQ ID NO: 71), UGAGGGGAGA (SEQ ID NO: 73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO: 74), CGGGUGGGAACACGUUUUUC (SEQ ID NO: 75), GACAGGUUUGGCCCUAUCCU (SEQ ID NO: 76), GAUCGUCAGCGCCGAGGCCU (SEQAC NO: 77) 78), UGAGGGUCUCGGCCACCUUC (SEQ ID NO: 79), AGGCUUCUACCCCGACCACG (SEQ ID NO: 80), CCGACCACGUGGAGCUGAGC (SEQ ID NO: 81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO: 83), SEAGGGGGA SEQ ID NO: 84), GCGCUGACGAUCUGGGUGAC (SEQ ID NO: 85), UGAGGGCGGGCUGCUCCUUG (SEQ ID NO: 86), GUUGCGGGGGUUCUGCCAGA (SEQ ID NO: 87), AGCUCAGCUCCACGUGGUCG (SEQ ID NO: G) CGGCUGCUCAGGCAGUAUC (SEQ ID NO: 89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO: 90), UGGCUCAAACACAGCGACCU (SEQ ID NO: 91),

ACUGGACUUGACAGCGGAAG (SEQ ID NO: 92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO: 93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO: 94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO: 95), CUCGGGGCQGA (SEC ID: 95), : 97), CCGAGAGCCCGUAGAAC (SEQ ID NO: 98), CCAGAUCGUCAGCGCCG (SEQ ID NO: 99), GAAUGACGAGUGGACCC (SEQ ID NO: 100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 101), GGUGACGUGA (SEQ ID NO: 101), GGUGUGGU (SEQ ID NO: 103), GACAGGUUUGGCCCUAUC (SEQ ID NO: 104), GAUACUGCCUGAGCAGCCGCCU (SEQ ID NO: 105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO: 106), GUGGAGCUGAGCUGGUGG (SEQ ID NO: 107) 108), GGCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 109), GCGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 110), GGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 111), GGCUGCUCCUGAGGGGGGG: SEQ ID NO: 114), GUGAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 115) and GAAUGGGAAGGAGGUGCACAG (SEQ ID NO: 116).

[001076] 253. The manufacturing article of modality 252, in which the gRNA has a targeting domain that comprises the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63).

[001077] 254. A kit comprising an article of manufacture of any of the modalities 234 to 253, and instructions for use.

[001078] 255. The modality kit 254, in which the instructions specify that said one or more agents and the polynucleotide are introduced into the cell.

[001079] 256. The modality kit 254 or 255, in which the instructions specify that said one or more agents and the polynucleotide are introduced simultaneously or sequentially, in any order.

[001080] 257. The kit of any of the modalities 254 to 257, in which the instructions specify that the introduction of the polynucleotide is carried out after the introduction of said one or more agents.

[001081] 258. The 257 modality kit, in which the instructions specify that the polynucleotide is introduced immediately after, or within about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of one or more agents capable of inducing a genetic disruption. X. EXAMPLES

[001082] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention. Example 1: Generation of Modified T Cells Expressing a Recombinate TCR

[001083] T cells are modified to contain genetic disruptions of the endogenous TCRα siding loci (TRAC) and β TCR constant gene (TRBC) and modified to express a recombinant T cell receptor (TCR) by targeted integration of the polynucleotide that encodes the recombinant TCR at the endogenous TRAC locus. A. Recombinant TCR Transgene Constructions

[001084] Polynucleotide constructs encoding an exemplary recombinant TCR that recognizes an epitope of human papilloma virus (HPV) E7 oncoprotein 16 were generated that contains a full-chain TCRβ sequence and a partial TCRα chain sequence separated by a sequence 2A ribosome jump. The partial recombinant TCRα coding sequence included a complete TCRα variable region sequence and a partial recombinant TCRα constant region sequence corresponds to a 5 'part of exon 1 of the TRAC gene (mentioned in SEQ ID NO: 142 ).

[001085] The sequences encoding recombinant TCRs have also been codon-optimized and / or modified by mutations to promote the formation of a non-native disulfide bond at the interface between the TCR constant domains to increase TCR pairing and stability. Non-native disulfide bonding was promoted by modifying the TCR chains at residue 48 in exon 1 of the constant region (Cα) of the TCRα chain from Thr to Cys and residue 57 of the constant region (Cβ) of the TCRβ chain from Ser to Cys (see Kuball et al. (2007) Blood, 109: 2331-2338). The construct was designed to produce an mRNA that encodes complete TCRβ and TCRα chains by incorporating the partial recombinant TCRα exon 1 sequence, including the sequence encoding a modified Cys residue, into exon 1 of the endogenous TRAC gene, so that the sequence of exon 1 of the endogenous TRAC gene is in structure with the partial recombinant TCRα coding sequence.

[001086] The sequences encoding the recombinant TCR were placed under the operable control of the human extension factor 1 alpha promoter (EF1α) (sequence mentioned in SEQ ID NO: 127), the MND promoter (sequence mentioned in SEQ ID NO: 126), which is a synthetic promoter that contains the U3 region of a MoMuLV LTR modified with myeloproliferative sarcoma virus enhancer, or a P2A ribosome bounce element (sequence mentioned in SEQ ID NO: 6) followed by sequences encoding the TCR re-

combining agent to direct the expression of the recombinant TCR of the endogenous TCRα promoter after targeted HDR-mediated integration into the human TRAC gene.

[001087] For HDR-directed integration, a construct of the adeno-associated virus (AAV) vector was generated that contains the exemplary recombinant TCR transgene flanked at the 5 'and 3' ends by 600 bp homology arms that were homologous to the sequence surrounding the target integration site and for transgene integration to produce a fusion mRNA in structure (5 'sequence homology arm mentioned in SEQ ID NO: 124; 3' sequence homology arm) SEQ ID NO: 125).

[001088] Polynucleotides encoding complete TCRβ and TCRα chains of the exemplary TCR sequence that separated by P2A ribosomal jump sequences were also generated. These polynucleotides contained an SV40 poly A region placed 3 'from the sequence encoding a complete TCRα chain and were flanked on each side by 600 bp homology arms that were homologous to the sequences surrounding the genetic break in the TRAC gene.

[001089] AAV stocks were produced by triple transfection of an AAV vector that included polynucleotides encoding an exemplary recombinant TCR, serotype-6 helper plasmids, and adenoviral helper in a 293T-17 cell line. Transfected cells were collected and lysed, and AAV stock was collected for cell transduction. B. Generation of Modified T Cells

[001090] Primary human T cells CD4 + and CD8 + were isolated by immunoaffinity selection of human peripheral sage mononuclear cells (PBMCs) obtained from healthy donors. The resulting cells were stimulated for 72 hours by culture with an anti-CD3 / anti-CD28 reagent at a 1: 1 ratio of cell to bead in medium containing human serum, IL-2 (100 U / mL), IL- 7 (10 ng / ml) and IL-15 (5 ng / ml) at 37ºC. After stimulation, CD3 / CD28 beads were removed magnetically and the cells were washed with PBS before electroporation.

[001091] To eliminate the endogenous TCR genes, the cells were electroporated with ribonucleoprotein (RNP) complexes that contain a guide RNA (gRNA) designed to target the site within exon 1 of the TCRα constant region gene (sequence targeting site for TRAC mentioned in SEQ ID NO: 117 and a consensus target site sequence common to exon 1 of both regions 1 and 2 constant TCR β (target site sequence for TRBC mentioned in SEQ ID NO: 118) and the protein Streptococcus pyogenes Cas9. The cells were then cultured in the medium containing human serum and IL-2 (50 U / mL), IL-7 (5 ng / mL) and IL-15 (0.5 ng / mL).

[001092] For targeted integration of the recombinant TCR into the TRAC locus, the cells were transduced with the appropriate AAV preparations described above, approximately 2 hours after RNP electroporation. The cells were subsequently expanded in culture medium containing cytokines. Cells subjected to transfection by simulation were used as controls. Example 1: Evaluation of excretion and function of recombinant TCR

[001093] T cells were modified to express the exemplary recombinant anti-HPV TCR 16 E7 as described in Example 1. On day 7 after transduction, cells were analyzed by flow cytometry for antibody staining for cell surface markers that included CD8 , an anti-Vbeta22 antibody specific for recombinant TCR, and with a peptide-MHC tetramer complexed with a recognized recombinant TCR, HPV 16 E7 peptide (11-19) (sequence mentioned in SEQ ID NO: 143) .

[001094] As shown in FIGS. 1A and 1B, HDR-mediated knock-in of recombinant TCR and elimination of endogenous TCRα and TCRβ resulted in TCR expression as indicated by the presence of CD8 + cells that express Vbeta22 (FIG. 1A) and bond to the HPV16 tetramer E7 (11-19) -MHC (FIG. 1B), when compared to transfection controls by simulation. Similar expression of the exemplary recombinant TCR was observed in cells after transduction with AAV that encodes total matching TCRβ and TCRα chains (called SV40 pA), under the control of the EF1α or MND promoter, as with AAV that encodes a recombinant TCRβ total and fusion of recombinant and endogenous TCRα chain sequences (called 3 'arm) under the control of the endogenous TRAC promoter (called P2A) or MND promoter.

[001095] Cytolytic activity was evaluated by incubating CD8 + effector cells expressing recombinant TCR with HPV-16 positive squamous cell carcinoma lineage, SCC-152, in a 5: 1 effector-to-target (E: T) ratio. The target cells were marked with Nu- cLight Red (NLR) to allow tracking by fluorescent microscopy. As controls, cocultures of target cells with CD8 + cells expressing a reference TCR capable of binding to HPV 16 E7 but containing mouse Cα and Cβ regions were analyzed ("E7 Ref mouse") or transfected by simulation. As shown in FIG. 2, cytolytic activity of cells expressing the exemplary recombinant TCR, generated by transduction with AAV encoding sequences of total recombinant TCRβ and TCRα chains (designated SV40 pA), under the control of the MND promoter or the endogenous TRAC promoter (designated P2A ); with AAV encoding a total recombinant TCRβ and fusion of recombinant and endogenous TCRα chain sequences (called the 3 'arm) under the control of the endogenous TRAC promoter or MND promoter were generally similar, and generally similar to the activity cytolytics of the reference TCR which contains a mouse constant region. Example 3: Evaluation of Homology Arm Lengths for Efficient HDR-Mediated Integration of Transgenic Sequences

[001096] Polynucleotides containing an exemplary transgene, flanked by varying lengths of homology arms were introduced into T cells for targeted integration of the transgene through homology-dependent repair (HDR), and the integration efficiency was estimated. A. Recombinant Transgene Constructions and Modified T Cell Generation

[001097] Exemplary HDR standard polynucleotides containing a transgene sequence encoding a green fluorescent protein (GFP), flanked by varying lengths of homology arms for targeted integration into the human TCRα constant region (TRAC) locus were introduced into cells T. Specifically, for HDR integration, AAV preparations containing standard nucleotide constructs encoding GFP were substantially generated as described in Example 1, except with the following differences: AAV constructs were generated that contain a polynucleotide. otid that encodes GFP under the operable control of the MND promoter and linked to a SV40 poly (A) sequence, flanked by 50, 100, 200, 300, 400, 500, or 600 base pairs of 5 'and 3' homology arms (SEQ ID NOS: 229-235 and 236-242, respectively) homologous to sequences surrounding the target integration site in the human TCRα constant region (TRAC) gene. The total length of the AAV construct was made constant using DNA load sequences between the SV40 poly (A) sequence and the 3 'homology arm.

[001098] For HDR-directed integration, primary human CD4 + and CD8 + T cells from four human donors (1 to 4 donor-

res) were combined in a 1: 1 ratio, stimulated and subjected to electroporation with ribonucleoprotein complexes (RNP) containing TRAC targeting gRNA, generally as described in Examples 1 to 3. After electroporation, cells were transduced - with AAV preparations containing the HDR standard polynucleotides which contain the various homology arm lengths described above. GFP expression was measured by flow cytometry at 24, 48, 72, 96 hours and 7 days after transduction with AAV, to determine an integration relationship (representing the percentage of total AAV within cells that integrated into the genome) with based on the following formula:. The high% of MFI and low% of MFI indicate the percentage of cells that were above or below a borderline MFI by flow cytometry, representing cells containing integrated GFP transgene or non-integrated AAV construction, respectively. Changes in the integration ratio with an increase in the length of the homology arm were assessed by subtracting the integration ratio from the next shortest arm length to the integration ratio of a particular arm member. B. Integration Relationship Assessment

[001099] As shown in FIGS. 3A-3B, a constant or increased integration ratio was observed at various time points assessed for each homology arm length, consistent with non-integrated AAV constructions diluting over time. Evaluation of changes in GFP expression patterns over time showed that the percentage of cells with integrated GFP transgene (high MFI) generally remained constant after 72 hours, but the percentage of cells containing AAV not only integrated (low MFI) continued to reduce.

[001100] As shown in FIGS. 4A-4B, the most substantial gains

Tanks in relation to integration seemed to occur at 200 bp of arm of homology, with smaller gauges at 300 bp. No substantial gain was observed between 300 bp and 500 bp, and an increase in the integration ratio was observed between 500 bp and 600 bp, in all donors. The results support the use of homology arms with a minimum of 300 bp for high integration efficiency, with homology arms with 600 bp providing even greater integration efficiency.

[001101] The present invention is not intended to be limited in scope to the particular described modalities, which are provided, for example, to illustrate various aspects of the invention. Various modifications to the described compositions and methods will become evident from the description and teachings contained in this document. Such variations can be practiced without departing from the real scope and spirit of the invention and are intended to be included in the scope of this invention.

Sequences SEQUENCE # 1 LEC atatccagaaccctgaccctgccgtgtaccagctgagagactcta aatccagtgacaagtctgtctgcctattcaccgattttgattctcaaa alpha constant TCR human caaatgtgtcacaaagtaaggattctgatgtgtatatcacagaca aaactgtgctagacatgaggtctatggacttcaagagcaacagt (TRAC) gctgtggcctggagcaacaaatctgactttgcatgtgcaaacgcc ttcaacaacagcattattccagaagacaccttcttccccagccca Sequence NCBI Reference ggtaagggcagctttggtgccttcgcaggctgtttccttgcttcagg: aatggccaggttctgcccagagctctggtcaatgatgtctaaaact NG_001332.3, cctctgattggtggtctcggccttatccattgccaccaaaaccctctt TRAC tttactaagaaacagtgagccttgttctggcagtccagagaatgac acgggaaaaaagcagatgaagagaaggtggcaggagaggg cacgtggcccagcctcagtctctccaactgagttcctgcctgcctg cctttgctcagactgtttgccccttactgctcttctaggcctcattctaa gccccttctccaagttgcctctccttatttctccctgtctgccaaaaaa tctttcccagctcactaagtcagtctcacgcagtcactcattaaccc accaatcactgattgtgccggcacatgaatgcaccaggtgttgaa gtggaggaattaaaaagtcagatgaggggtgtgcccagaggaa gcaccattctagttgggggagcccatctgtcagctgggaaaagtc caaataacttcagattggaatgtgttttaactcagggtt gagaaaa cagctaccttcaggacaaaagtcagggaagggctctctgaaga aatgctacttgaagataccagccctaccaagggcagggagagg accctatagaggcctgggacaggagctcaatgagaaaggaga agagcagcaggcatgagttgaatgaaggaggcagggccgggt cacagggccttctaggccatgagagggtagacagtattctaagg acgccagaaagctgttgatcggcttcaagcaggggagggacac ctaatttgcttttcttttttttttttttttttttttttttttttgagatggagttttgctct tgttgcccaggctggagtgcaatggtgcatcttggctcactgcaac ctccgcctcccaggttcaagtgattctcctgcctcagcctcccgagt agctgagattacaggcacccgccaccatgcctggctaattttttgt atttttagtagagacagggtttcactatgttggccaggctggtctcg aactcctgacctcaggtgatccacccgcttcagcctcccaaagtg ctgggattacaggcgtgagccaccacacccggcctgcttttcttaa agatcaatctgagtgctgtacggagagtgggttgtaagccaaga gtagaagcagaaagggagcagttgcagcagagagatgatgga ggcctgggcagggtggtggcagggaggtaaccaacaccattca

SEQUENCE # LEC ggtttcaaaggtagaaccatgcagggatgagaaagcaaagag gggatcaaggaaggcagctggattttggcctgagcagctgagtc aatgatagtgccgtttactaagaagaaaccaaggaaaaaatttg gggtgcagggatcaaaactttttggaacatatgaaagtacgtgttt atactctttatggcccttgtcactatgtatgcctcgctgcctccattgg actctagaatgaagccaggcaagagcagggtctatgtgtgatgg cacatgtggccagggtcatgcaacatgtactttgtacaaacagtgt atattgagtaaatagaaatggtgtccaggagccgaggtatcggtc ctgccagggccaggggctctccctagcaggtgctcatatgctgta agttccctccagatctctccacaaggaggcatggaaaggctgta gttgttcacctgcccaagaactaggaggtctggggtgggagagtc agcctgctctggatgctgaaagaatgtctgtttttccttttagaaagtt cctgtgatgtcaagctggtcgagaaaagctttgaaacaggtaag acaggggtctagcctgggtttgcacaggattgcggaagtgatga acccgcaataaccctgcctggatgagggagtgggaagaaatta gtagatgtgggaatgaatgatgaggaatggaaacagcggttca agacctgcccagagctgggtggggtctctcctgaatccctctcac catctctgactttccattctaagcactttgaggatgagtttctagcttc aatagaccaaggactctctcctaggcctctgtattcctttcaacagc tccactgtcaagagagccagagagagcttctgggtggcccagct gtgaaatttctgagtcccttagggatagccctaaacgaaccagat catcctgagg acagccaagaggttttgccttctttcaagacaagc aacagtactcacataggctgtgggcaatggtcctgtctctcaaga atcccctgccactcctcacacccaccctgggcccatattcatttcc atttgagttgttcttattgagtcatccttcctgtggtagcggaactcact aaggggcccatctggacccgaggtattgtgatgataaattctgag cacctaccccatccccagaagggctcagaaataaaataagagc caagtctagtcggtgtttcctgtcttgaaacacaatactgttggccct ggaagaatgcacagaatctgtttgtaaggggatatgcacagaag ctgcaagggacaggaggtgcaggagctgcaggcctcccccac ccagcctgctctgccttggggaaaaccgtgggtgtgtcctgcagg ccatgcaggcctgggacatgcaagcccataaccgctgtggcctc ttggttttacagatacgaacctaaactttcaaaacctgtcagtgattg ggttccgaatcctcctcctgaaagtggccgggtttaatctgctcatg acgctgcggctgtggtccagctgaggtgaggggccttgaagctg ggagtggggtttagggacgcgggtctctgggtgcatcctaagctct gagagcaaacctccctgcagggtcttgcttttaagtccaaagcct

SEQUENCE # LEC gagcccaccaaactctcctacttcttcctgttacaaattcctcttgtg caataataatggcctgaaacgctgtaaaatatcctcatttcagccg cctcagttgcacttctcccctatgaggtaggaagaacagttgtttag aaacgaagaaactgaggccccacagctaatgagtggaggaa gagagacacttgtgtacaccacatgccttgtgttgtacttctctcacc gtgtaacctcctcatgtcctctctccccagtacggctctcttagctca gtagaaagaagacattacactcatattacaccccaatcctggcta gagtctccgcaccctcctcccccagggtccccagtcgtcttgctga caactgcatcctgttccatcaccatcaaaaaaaaactccaggctg ggtgcgggggctcacacctgtaatcccagcactttgggaggcag aggcaggaggagcacaggagctggagaccagcctgggcaac acagggagaccccgcctctacaaaaagtgaaaaaattaacca ggtgtggtgctgcacacctgtagtcccagctacttaagaggctga gatgggaggatcgcttgagccctggaatgttgaggctacaatga gctgtgattgcgtcactgcactccagcctggaagacaaagcaag atcctgtctcaaataataaaaaaaataagaactccagggtacatt tgctcctagaactctaccacatagccccaaacagagccatcacc atcacatccctaacagtcctgggtcttcctcagtgtccagcctgact tctgttcttcctcattccagatctgcaagattgtaagacagcctgtgc tccctcgctccttcctctgcattgcccctcttctccctctccaaacaga gggaactctcctacccccaaggaggtgaaagctgctaccacctc tgtgcc cccccggcaatgccaccaactggatcctacccgaattta tgattaagattgctgaagagctgccaaacactgctgccaccccct ctgttcccttattgctgcttgtcactgcctgacattcacggcagaggc aaggctgctgcagcctcccctggctgtgcacattccctcctgctcc ccagagactgcctccgccatcccacagatgatggatcttcagtgg gttctcttgggctctaggtcctgcagaatgttgtgaggggtttattttttt ttaatagtgttcataaagaaatacatagtattcttcttctcaagacgt ggggggaaattatctcattatcgaggccctgctatgctgtgtatctg ggcgtgttgtatgtcctgctgccgatgccttc 2 aggacctgaacaaggtgttcccacccgaggtcgctgtgtttgagc catcagaagcagagatctcccacacccaaaaggccacactggt beta TCR constant human gtgcctggccacaggcttcttccccgaccacgtggagctgagctg 1 gtgggtgaatgggaaggaggtgcacagtggggtcagcacaga (TRBC1) cccgcagcccctcaaggagcagcccgccctcaatgactccaga tactgcctgagcagccgcctgagggtctcggccaccttctggcag Reference Sequence

SEQUENCE # LEC aacccccgcaaccacttccgctgtcaagtccagttctacgggctct NCBI: cggagaatgacgagtggacccaggatagggccaaacccgtca NG_001333.2, cccagatcgtcagcgccgaggcctggggtagagcaggtgagtg TRBC1 gggcctggggagatgcctggaggagattaggtgagaccagcta ccagggaaaatggaaagatccaggtagcagacaagactagat ccaaaaagaaaggaaccagcgcacaccatgaaggagaattg ggcacctgtggttcattcttctcccagattctcagcccaacagagc caagcagctgggtcccctttctatgtggcctgtgtaactctcatctgg gtggtgccccccatccccctcagtgctgccacatgccatggattgc aaggacaatgtggctgacatctgcatggcagaagaaaggaggt gctgggctgtcagaggaagctggtctgggcctgggagtctgtgcc aactgcaaatctgactttacttttaattgcctatgaaaataaggtctct catttattttcctctccctgctttctttcagactgtggctttacctcgggta agtaagcccttccttttcctctccctctctcatggttcttgacctagaa ccaaggcatgaagaactcacagacactggagggtggagggtg ggagagaccagagctacctgtgcacaggtacccacctgtccttc ctccgtgccaacagtgtcctaccagcaaggggtcctgtctgccac catcctctatgagatcctgctagggaaggccaccctgtatgctgtg ctggtcagcgcccttgtgttgatggccatggtaagcaggagggca ggatggggccagcaggctggaggtgacacactgacaccaagc acccagaagtatagagtccctgccaggattggag ctgggcagta gggagggaagagatttcattcaggtgcctcagaagataacttgc acctctgtaggatcacagtggaagggtcatgctgggaaggaga agctggagtcaccagaaaacccaatggatgttgtgatgagcctta ctatttgtgtggtcaatgggccctactactttctctcaatcctcacaac tcctggctcttaataacccccaaaactttctcttctgcaggtcaaga gaaaggatttctga 3 aggacctgaaaaacgtgttcccacccgaggtcgctgtgtttgagc catcagaagcagagatctcccacacccaaaaggccacactggt beta constant human atgcctggccacaggcttctaccccgaccacgtggagctgagct TCR 2 ggtgggtgaatgggaaggaggtgcacagtggggtcagcacag (TRBC2) acccgcagcccctcaaggagcagcccgccctcaatgactccag atactgcctgagcagccgcctgagggtctcggccaccttctggca Sequence NCBI Reference gaacccccgcaaccacttccgctgtcaagtccagttctacgggct: ctcggagaatgacgagtggacccaggatagggccaaacccgt NG_001333.2, cacccagatcgtcagcgccgaggcctggggtagagcaggtga TRBC2

SEQUENCE # LEC gtggggcctggggagatgcctggaggagattaggtgagaccag ctaccagggaaaatggaaagatccaggtagcggacaagacta gatccagaagaaagccagagtggacaaggtgggatgatcaag gttcacagggtcagcaaagcacggtgtgcacttcccccaccaag aagcatagaggctgaatggagcacctcaagctcattcttccttca gatcctgacaccttagagctaagctttcaagtctccctgaggacca gccatacagctcagcatctgagtggtgtgcatcccattctcttctgg ggtcctggtttcctaagatcatagtgaccacttcgctggcactgga gcagcatgagggagacagaaccagggctatcaaaggaggct gactttgtactatctgatatgcatgtgtttgtggcctgtgagtctgtgat gtaaggctcaatgtccttacaaagcagcattctctcatccatttttctt cccctgttttctttcagactgtggcttcacctccggtaagtgagtctct cctttttctctctatctttcgccgtctctgctctcgaaccagggcatgg agaatccacggacacaggggcgtgagggaggccagagccac ctgtgcacaggtgcctacatgctctgttcttgtcaacagagtcttacc agcaaggggtcctgtctgccaccatcctctatgagatcttgctagg gaaggccaccttgtatgccgtgctggtcagtgccctcgtgctgatg gccatggtaaggaggagggtgggatagggcagatgatggggg caggggatggaacatcacacatgggcataaaggaatctcaga gccagagcacagcctaatatatcctatcacctcaatgaaaccata atgaagccagactggggagaaaatgcagggaatatcacagaa tgcatcatggga ggatggagacaaccagcgagccctactcaaa ttaggcctcagagcccgcctcccctgccctactcctgctgtgccat agcccctgaaaccctgaaaatgttctctcttccacaggtcaagag aaaggattccagaggctag 4 cgtgaggctccggtgcccgtcagtgggcagagcgcacatcgcc cacagtccccgagaagttggggggaggggtcggcaattgaacc EF1alpha promoter ggtgcctagagaaggtggcgcggggtaaactgggaaagtgatg (GenBank: J04617.1 tcgtgtactggctccgcctttttcccgagggtgggggagaaccgta) tataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgc cgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctg gcctctttacgggttatggcccttgcgtgccttgaattacttccacgc ccctggctgcagtacgtgattcttgatcccgagcttcgggttggaa gtgggtgggagagttcgaggccttgcgcttaaggagccccttcgc ctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcg tgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagt

SEQUENCE # LEC ctctagccatttaaaatttttgatgacctgctgcgacgctttttttctgg caagatagtcttgtaaatgcgggccaagatctgcacactggtattt cggtttttggggccgcgggcggcgacggggcccgtgcgtcccag cgcacatgttcggcgaggcggggcctgcgagcgcggccaccg agaatcggacgggggtagtctcaagctggccggcctgctctggt gcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaa ggctggcccggtcggcaccagttgcgtgagcggaaagatggcc gcttcccggccctgctgcagggagctcaaaatggaggacgcgg cgctcgggagagcgggcgggtgagtcacccacacaaaggaa aagggcctttccgtcctcagccgtcgcttcatgtgactccacggag taccgggcgccgtccaggcacctcgattagttctcgagcttttgga gtacgtcgtctttaggttggggggaggggttttatgcgatggagtttc cccacactgagtgggtggagactgaagttaggccagcttggcac ttgatgtaattctccttggaatttgccctttttgagtttggatcttggttca ttctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtg tcgtgaa 5 CGTGAGGCTCCGGTGCCCGTCAGTGGGCAG Promoter AGCGCACATCGCCCACAGTCCCCGAGAAGT EF1alpha

TGGGGGGAGGGGTCGGCAATTGAACCGGTG CCTAGAGAAGGTGGCGCGGGGTAAACTGGG AAAGTGATGTCGTGTACTGGCTCCGCCTTTT TCCCGAGGGTGGGGGAGAACCGTATATAAG TGCACTAGTCGCCGTGAACGTTCTTTTTCGC AACGGGTTTGCCGCCAGAACACAGGTAAGT GCCGTGTGTGGTTCCCGCGGGCCTGGCCTC TTTACGGGTTATGGCCCTTGCGTGCCTTGAA TTACTTCCACCTGGCTGCAGTACGTGATTCT TGATCCCGAGCTTCGGGTTGGAAGTGGGTG GGAGAGTTCGTGGCCTTGCGCTTAAGGAGC CCCTTCGCCTCGTGCTTGAGTTGTGGCCTG GCCTGGGCGCTGGGGCCGCCGCGTGCGAA TCTGGTGGCACCTTCGCGCCTGTCTCGCTG CTTTCGATAAGTCTCTAGCCATTTAAAATTTT TGATGACCTGCTGCGACGCTTTTTTTCTGGC AAGATAGTCTTGTAAATGCGGGCCAAGATCA GCACACTGGTATTTCGGTTTTTGGGGCCGCG # SEQUENCES NOTE GGCGGCGACGGGGCCCGTGCGTCCCAGCG CACATGTTCGGCGAGGCGGGGCCTGCGAGC GCGGCCACCGAGAATCGGACGGGGGTAGTC TCAAGCTGCCCGGCCTGCTCTGGTGCCTGG CCTCGCGCCGCCGTGTATCGCCCCGCCCTG GGCGGCAAGGCTGGCCCGGTCGGCACCAG TTGCGTGAGCGGAAAGATGGCCGCTTCCCG GCCCTGCTGCAGGGAGCACAAAATGGAGGA CGCGGCGCTCGGGAGAGCGGGCGGGTGAG TCACCCACACAAAGGAAAAGGGCCTTTCCGT CCTCAGCCGTCGCTTCATGTGACTCCACGGA GTACCGGGCGCCGTCCAGGCACCTCGATTA GTTCTCCAGCTTTTGGAGTACGTCGTCTTTA GGTTGGGGGGAGGGGTTTTATGCGATGGAG TTTCCCCACACTGAGTGGGTGGAGACTGAA GTTAGGCCAGCTTGGCACTTGATGTAATTCT CCTTGGAATTTGCCCTTTTTGAGTTTGGATCT TGGTTCATTCTCAAGCCTCAGACAGTGGTTC AAAGTTTTTTTCTTCCATTTCAGGTGTCGTGA

AAACTACCCCTAAAAGCCAAA 6 LEGGGEGRGSLLTCGDVEENPGPR T2A 7 EGRGSLLTCGDVEENPGP T2A 8 GSGATNFSLLKQAGDVEENPGP P2A 9 ATNFSLLKQAGDVEENPGP P2A 10 QCTNYALLKLAGDVESNQPLFRPFGFLPDFNYLVLVLVLVLVLQFNYLPHE2EYEYEYEYEYEYEYEYEYEYE

KDSLSINATNIKHFKNCTSISGDLHILPVAFRGD SFTHTPPLDPQELDILKTVKEITGFLLIQAWPEN RTDLHAFENLEIIRGRTKQHGQFSLAVVSLNITS LGLRSLKEISDGDVIISGNKNLCYANTINWKKLF GTSGQKTKIISNRGENSCKATGQVCHALCSPE GCWGPEPRDCVSCRNVSRGRECVDKCNLLE GEPREFVENSECIQCHPECLPQAMNITCTGRG PDNCIQCAHYIDGPHCVKTCPAGVMGENNTLV WKYADAGHVCHLCHPNCTYGCTGPGLEGCPT # SEQUENCES NOTE

NGPKIPSIATGMVGALLLLLVVALGIGLFM 13 RKVCNGIGIGEFKDSLSINATNIKHFKNCTSISG EGFRt

DLHILPVAFRGDSFTHTPPLDPQELDILKTVKEI TGFLLIQAWPENRTDLHAFENLEIIRGRTKQHG QFSLAVVSLNITSLGLRSLKEISDGDVIISGNKN LCYANTINWKKLFGTSGQKTKIISNRGENSCKA TGQVCHALCSPEGCWGPEPRDCVSCRNVSR GRECVDKCNLLEGEPREFVENSECIQCHPECL PQAMNITCTGRGPDNCIQCAHYIDGPHCVKTC PAGVMGENNTLVWKYADAGHVCHLCHPNCTY GCTGPGLEGCPTNGPKIPSIATGMVGALLLLLV

VALGIGLFM 14 DIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQIN Alpha Constant VPKTMESGTFITDKTVLDMKAMDSKSNGAIAW TCR from SNQTSFTCQDIFKETNATYPSSDVPCDATLTEK mouse

SFETDMNLNFQNLSVMGLRILLLKVAGFNLLMT

LRLWSS 15 NIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQIN Alpha VPKTMESGTFITDKTVLDMKAMDSKSNGAIAW TCR SNQTSFTCQDIFKETNATYPSSDVPCDATLTEK mouse mouse

SFETDMNLNFQNLSVMGLRILLLKVAGFNLLMT

LRLWSS 16 EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLA constant RGFFPDHVELSWWVNGKEVHSGVSTDPQAYK beta TCR of mouse ESNYSYCLSSRLRVSATFWHNPRNHFRCQVQ FHGLSEEDKWPEGSPKPVTQNISAEAWGRAD (CGITSASYQQGVLSATILYEILLGKATLYAVLVS UniProt P01852)

TLVVMAMVKRKNS 17 DLRNVTPPKVSLFEPSKAEIANKQKATLVCLAR Beta constant GFFPDHVELSWWVNGKEVHSGVSTDPQAYKE of SNYSYCLSSRLRVSATFWHNPRNHFRCQVQF mouse

HGLSEEDKWPEGSPKPVTQNISAEAWGRADC GITSASYHQGVLSATILYEILLGKATLYAVLVSG

LVLMAMVKRKNS 18 gggtctctctggttagaccagatctgagcctgggagctctctggct MND promoter aactagggaacccactgcttaagcctcaataaagcttgccttgag

# 19 SEQUENCE ANNOTATION tgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactaga gatccctcagacccttttagtcagtgtggaaaatctctagca PNIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQ alpha TNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV human TCR constant AWSNKSDFACANAFNNSIIPEDTFFPSPESSCD VKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF (NLLMTLRLWSS UniProt P01848) beta-20 Constant EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA TGFFPDHVELSWWVNGKEVHSGVSTDPQPLK human TCR EQPALNDSRYCLSSRLRVSATFWQNPRNHFR 1 CQVQFYGLSENDEWTQDRAKPVTQIVSAEAW (GRADCGFTSVSYQQGVLSATILYEILLGKATLY UniProt P01850)

AVLVSALVLMAMVKRKDF 21 DLKNVFPPEVAVFEPSEAEISHTQKATLVCLAT GFYPDHVELSWWVNGKEVHSGVSTDPQPLKE TCR beta constant human QPALNDSRYCLSSRLRVSATFWQNPRNHFRC 2 QVQFYGLSENDEWTQDRAKPVTQIVSAEAWG (UniProt RADCGFTSESYQQGVLSATILYEILLGKATLYA A0A5B9)

VLVSALVLMAMVKRKDSRG 22 -PGGG- (SGGGG) NP-wherein P is proline, G is glycine and S linker is Linker 24 GSADDAKKDAAKKDGKS serine 23 NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Realize human TCR alpha NVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV (Genbank Accession No. CAA26636.1 KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN of LLMTLRLWSS Access) 25 EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA TGFYPDHVELSWWVNGKEVHSGVSTDPQPLK constant beta TCR human EQPALNDSRYCLSSRLRVSATFWQNPRNHFR (No. CQVQFYGLSENDEWTQDRAKPVTQIVSAEAW of GRADCGFTSESYQQGVLSATILYEILLGKATLY UniProt AVLVSALVLMAMVKRKDSRG A0A0G2JNG9 Access) 26 AGCGCTCTCGTACAGAGTTGGCATTATAATA Sequence

# SEQUENCES ANNOTATION CGACTCACTATAGGGGAGAATCAAAATCGGT transcription GAATGTTTTAGAGCTAGAAATAGCAAGTTAAA TRAC gRNA

ATAAGGCTAGTCCGTTATCAACTTGAAAAAG

TGGCACCGAGTCGGTGCTTTTTTT 27 GAGAAUCAAAAUCGGUGAAUGUUUUAGAGC Sequence UAGAAAUAGCAAGUUAAAAUAAGGCUAGUC TRAC gRNA

CGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGCUUUU 28 UCUCUCAGCUGGUACACGGC TRAC-10 gRNA targeting domain 29 UGGAUUUAGAGUCUCUCAGC TRAC-110 gRNA targeting domain 30 ACACGGCAGGGUCAGGGUUC TRAC-116 GAGAUCAILA 4 33 CUCAGCUGGUACACGGC TRAC-49 gRNA targeting domain 34 UGGUACACGGCAGGGUC gRNA targeting domain

# SEQUENCES ANNOTATION TRAC-2 35 GCUAGACAUGAGGUCUA TRAC-30 gRNA targeting domain 36 GUCAGAUUUGUUGCUCC trac-43 gRNA targeting domain 37 UCAGCUGGUACACGGCA TRAC-23 gRNA targeting domain 38 GCAGACAGACGGGGACGACGACGACGACGACGAC domain TRAC-25 gRNA targeting 40 CUUCAAGAGCAACAGUGCUG TRAC-128 gRNA targeting domain 41 AGAGCAACAGUGCUGUGGCC TRAC-105 gRNA targeting domain 42 AAAGUCAGAUUUGUUGCUCC TRAC-106 gRNA targeting domain 43 ACAAACUGUGU

# SEQUENCES TRAC-123 gRNA ANNOTATION 44 AAACUGUGCUAGACAUG TRAC-64 gRNA targeting domain 45 UGUGCUAGACAUGAGGUCUA TRAC-97 gRNA targeting domain 46 GGCUGGGGAAGAAGGUGUCUUC gRNA targeting domain TRACGA-148 47 gRNA targeting domain TRAC-234 49 GUUUUGUCUGUGAUAUACACAU gRNA targeting domain TRAC-167 50 GGCAGACAGACUUGUCACUGGAUU gRNA targeting domain TRAC-177 51 GCAGACAGACUUGUCACUGGAUU gRNA targeting domain TRAC-176 52 GACNA

# SEQUENCES ANNOTATION TRAC-257 gRNA targeting 53 GUGAAUAGGCAGACAGACUUGUCA TRAC-233 gRNA targeting domain 54 GAAUAGGCAGACAGACUUGUCA trac-231 gRNA targeting domain 55 GAGUCUCUCAGCUGGUGACACGGGACGGGACGGGACGGACAGGGGGACGGGGACGGGGACGGGGGGGGGACGGGGGGGGGGGGGGGACGGGGGACGGGGACGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGUC GGUACACGGCAGGGUCAGGGUU gRNA targeting domain TRAC-179 58 GUACACGGCAGGGUCAGGGUU gRNA targeting domain TRAC-178 59 CACCCAGAUCGUCAGCGCCG gRNA targeting domain TRBC-40 60 CAAACACAGCGACCUCGGCGGCUCGGGCU

# SEQUENCES ANNOTATION 61 UGACGAGUGGACCCAGGAUA TRBC-25 gRNA targeting domain 62 GGCUCUCGGAGAAUGACGAG TRBC-35 gRNA targeting domain 63 GGCCUCGGCGCUGACGAUCU TRBC-50 GRNA targeting domain TRAG-50 gRNA targeting domain TRBC-49 66 AGUCCAGUUCUACGGGCUCU gRNA targeting domain TRBC-51 67 CGCUGUCAAGUCCAGUUCUA gRNA targeting domain TRBC-26 68 AUCGUCAGCGCCGAGGCCUG gRNA targeting domain TRBC-47 69 UCAAACACAGGGACCGGCUCCGG

# SEQUENCES ANNOTATION TRBC-45 70 CGUAGAACUGGACUUGACAG TRBC-34 gRNA targeting domain 71 AGGCCUCGGCGCUGACGAUC TRBC-227 gRNA targeting domain 72 UGACAGCGGAAGUGGUUGCG TRBC-41 GUGGAGGGAGGAGGGAGGAGGAGGGAGGGAGGAGGAGGGAGGAGGGAGGAGGGAGGAGGAGGAGGGGGGGGGGGGGGGGGGGGGGGGGGC TRBC-206 gRNA targeting 75 CGGGUGGGAACACGUUUUUC TRBC-32 gRNA targeting domain 76 GACAGGUUUGGCCCUAUCCU TRBC-276 gRNA targeting domain 77 GAUCGUCAGCGCCGAGGCCU TRBC-274 gRNA targeting domain TRAC-274 78 GACCA

# SEQUENCE annotation gRNA TRBC-230 79 UGAGGGUCUCGGCCACCUUC gRNA targeting domain TRBC-235 80 AGGCUUCUACCCCGACCACG gRNA targeting domain TRBC-38 81 CCGACCACGUGGAGCUGAGC targeting domain gRNA TRBC-223 82 UGACAGGUUUGGCCCUAUCC targeting domain gRNA TRBC-221 83 CUUGACAGCGGAAGUGGUUG TRBC-48 gRNA targeting domain 84 AGAUCGUCAGCGCCGAGGCC TRBC-216 gRNA targeting domain 85 GCGCUGACGAUCUGGGUGAC TRBC-210 gRNA targeting domain 86 UGAGGGCGGGCUGCUCCUUG gRNA targeting domain TRBC-268

# SEQUENCE ANNOTATION targeting gRNA TRBC-193 88 AGCUCAGCUCCACGUGGUCG targeting domain gRNA TRBC-246 89 GCGGCUGCUCAGGCAGUAUC targeting domain gRNA TRBC-228 90 GCGGGGGUUCUGCCAGAAGG gRNA targeting domain TRBC-43 91 UGGCUCAAACACAGCGACCU targeting domain gRNA TRBC-272 92 ACUGGACUUGACAGCGGAAG TRBC-33 gRNA targeting domain 93 GACAGCGGAAGUGGUUGCGG TRBC-44 gRNA targeting domain 94 GCUGUCAAGUCCAGUUCUAC gRNA targeting domain TRBC-211 95 GUAUCUGGAGUCAUUGAGGGGBC targeting domain

# SEQUENCES ANNOTATION 96 CUCGGCGCUGACGAUCU TRBC-18 gRNA targeting domain 97 CCUCGGCGCUGACGAUC TRBC-6 gRNA targeting domain 98 CCGAGAGCCCGUAGAAC gRNA TRBC-85 targeting domain TRBC-93 101 GGGUGACAGGUUUGGCCCUAUC gRNA targeting domain TRBC-415 102 GGUGACAGGUUUGGCCCUAUC gRNA targeting domain TRBC-414 103 GUGACAGGUUUGGCCCUAUC gRNA targeting domain TRBC-310 104 GACAGGUUCGU

# SEQUENCE ANNOTATION TRBC-308 105 GAUACUGCCUGAGCAGCCGCCU gRNA targeting domain TRBC-401 106 GACCACGUGGAGCUGAGCUGGUGG targeting domain gRNA TRBC-468 107 GUGGAGCUGAGCUGGUGG targeting domain gRNA TRBC-462 108 GGGCGGGCUGCUCCUUGAGGGGCU targeting domain gRNA TRBC-424 109 GGCGGGCUGCUCCUUGAGGGGCU domain TRBC-423 gRNA targeting 110 GCGGGCUGCUCCUUGAGGGGCU TRBC-422 gRNA targeting domain 111 GGGCUGCUCCUUGAGGGGCU TRBC-420 gRNA targeting domain 112 GGCUGCUCCUUGAGGGGCU 4GRGGCUGGUGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG

# SEQUENCES TRBC-418 gRNA ANNOTATION 114 GGUGAAUGGGAAGGAGGUGCACAG TRBC-445 gRNA targeting domain 115 GUGAAUGGGAAGGAGGUGCACAG TRBC-444 gRNA targeting domain 116 GAAUGGGAAGGAGGUGCACAGTTTGGTACTRACTIONGATTTTRAGGTT

TRAC 118 AGATCGTCAGCGCCGAGGCC targeting sequence

TRBC 119 CTGACCTCTTCTCTTCCTCCCACAG Splicer site acceptor

HBB 120 121 TTTCTCTCCACAG IgG acceptor splice site PYIQNPEPAVYQLKDPRSQDSTLCLFTDFDSQI TCR alpha constant NVPKTMESGTFITDKTVLDMKAMDSKSNGAIA WSNQTSFTCQDIFKETNATYPSSDVPCDATLT mouse EKSFETDMNLNFQNLSVMGLRILLLKVAGFNLL (MTLRLWSS UniProt P01849) 122 IQNPEPAVYQLKDPRSQDSTLCLFTDFDSQINV constant alpha of PKTMESGTFITDKCVLDMKAMDSKSNGAIAWS NQTSFTCQDIFKETNATYPSSDVPCDATLTEKS modified TCR mice FETDMNLNFQNLLVIVLRILLLKVAGFNLLMTLR

# SEQUENCES NOTE

LWSS 123 EDLRNVTPPKVSLFEPSKAEIANKQKATLVCLA beta constant RGFFPDHVELSWWVNGKEVHSGVCTDPQAY from KESNYSYCLSSRLRVSATFWHNPRNHFRCQV mouse QFHGLSEEDKWPEGSPEA

DCGITSASYQQGVLSATILYEILLGKATLYAVLV

STLVVMAMVKRKNS 124 CTCTATCAATGAGAGAGCAATCTCCTGGTAA TGTGATAGATTTCCCAACTTAATGCCAACATA arm 5 'homology

CCATAAACCTCCCATTCTGCTAATGCCCAGC TRAC CTAAGTTGGGGAGACCACTCCAGATTCCAAG ATGTACAGTTTGCTTTGCTGGGCCTTTTTCC CATGCCTGCCTTTACTCTGCCAGAGTTATATT GCTGGGGTTTTGAAGAAGATCCTATTAAATA AAAGAATAAGCAGTATTATTAAGTAGCCCTG CATTTCAGGTTTCCTTGAGTGGCAGGCCAGG CCTGGCCGTGAACGTTCACTGAAATCATGGC CTCTTGGCCAAGATTGATAGCTTGTGCCTGT CCCTGAGTCCCAGTCCATCACGAGCAGCTG GTTTCTAAGATGCTATTTCCCGTATAAAGCAT GAGACCGTGACTTGCCAGCCCCACAGAGCC CCGCCCTTGTCCATCACTGGCATCTGGACTC CAGCCTGGGTTGGGGCAAAGAGGGAAATGA GATCATGTCCTAACCCTGATCCTCTTGTCCC ACAGATATCCAGAACCCTGACCCTGCCGTGT ACCAGCTGAGAGACTCTAAATCCAGTGACAA

GTCTGTCTGCCTATTCACCGAT 125 TTTGATTCTCAAACAAATGTGTCACAAAGTAA Arm of GGATTCTGATGTGTATATCACAGACAAAACT homology 3 ’

GTGCTAGACATGAGGTCTATGGACTTCAAGA TRAC GCAACAGTGCTGTGGCCTGGAGCAACAAAT CTGACTTTGCATGTGCAAACGCCTTCAACAA CAGCATTATTCCAGAAGACACCTTCTTCCCC AGCCCAGGTAAGGGCAGCTTTGGTGCCTTC GCAGGCTGTTTCCTTGCTTCAGGAATGGCCA GGTTCTGCCCAGAGCTCTGGTCAATGATGTC # SEQUENCES NOTE TAAAACTCCTCTGATTGGTGGTCTCGGCCTT ATCCATTGCCACCAAAACCCTCTTTTTACTAA GAAACAGTGAGCCTTGTTCTGGCAGTCCAGA GAATGACACGGGAAAAAAGCAGATGAAGAG AAGGTGGCAGGAGAGGGCACGTGGCCCAG CCTCAGTCTCTCCAACTGAGTTCCTGCCTGC CTGCCTTTGCTCAGACTGTTTGCCCCTTACT GCTCTTCTAGGCCTCATTCTAAGCCCCTTCT CCAAGTTGCCTCTCCTTATTTCTCCCTGTCTG CCAAAAAATCTTTCCCAGCTCACTAAGTCAG TCTCACGCAGTCACTCATTAACCCACCAATC

ACTGATTGTG 126 GAACAGAGAAACAGGAGAATATGGGCCAAA MND promoter

CAGGATATCTGTGGTAAGCAGTTCCTGCCCC GGCTCAGGGCCAAGAACAGTTGGAACAGCA GAATATGGGCCAAACAGGATATCTGTGGTAA GCAGTTCCTGCCCCGGCTCAGGGCCAAGAA CAGATGGTCCCCAGATGCGGTCCCGCCCTC AGCAGTTTCTAGAGAACCATCAGATGTTTCC AGGGTGCCCCAAGGACCTGAAATGACCCTG TGCCTTATTTGAACTAACCAATCAGTTCGCTT CTCGCTTCTGTTCGCGCGCTTCTGCTCCCCG AGCTCTATATAAGCAGAGCTCGTTTAGTGAA

CCGTCAGATC 127 GGATCTGCGATCGCTCCGGTGCCCGTCAGT Promoter GGGCAGAGCGCACATCGCCCACAGTCCCCG Ef1alfa with AGAAGTTGGGGGGAGGGGTCGGCAATTGAA enhancer CCGGTGCCTAGAGGAGGGGGGGA

ACTGGGAAAGTGATGTCGTGTACTGGCTCC GCCTTTTTCCCGAGGGTGGGGGAGAACCGT ATATAAGTGCAGTAGTCGCCGTGAACGTTCT TTTTCGCAACGGGTTTGCCGCCAGAACACAG CTGAAGCTTCGAGGGGCTCGCATCTCTCCTT CACGCGCCCGCCGCCCTACCTGAGGCCGCC ATCCACGCCGGTTGAGTCGCGTTCTGCCGC CTCCCGCCTGTGGTGCCTCCTGAACTGCGT # SEQUENCES NOTE CCGCCGTCTAGGTAAGTTTAAAGCTCAGGTC GAGACCGGGCCTTTGTCCGGCGCTCCCTTG GAGCCTACCTAGACTCAGCCGGCTCTCCAC GCTTTGCCTGACCCTGCTTGCTCAACTCTAC GTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTA

CAGATCCAAGCTGTGACCGGCGCCTAC 128 GGATCTGGAGCGACGAATTTTAGTCTACTGA AACAAGCGGGAGACGTGGAGGAAAACCCTG nucleotide sequence GACCT P2A 129 RVKFSRSAEPPAYQQGQNQLYNELNLGRREE

YDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR 130 RVKFSRSADAPAYKQGQNQLYNELNLGRREE CD3 zeta

YDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGL

STATKDTYDALHMQALPPR 131 ESKYGPPCPPCP spacer (IgG4 hinge) 132 GAATCTAAGTACGGACCGCCCTGCCCCCCTT GCCCT spacer (IgG4 hinge) 133 ESKYGPPCPPCPGQPREPQVYTLPPSQEEMT Articulação- KNQVSLTCLVKGFYPSDIAVEWESNGQPENNY spacer CH3

KTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVF

SCSVMHEALHNHYTQKSLSLSLGK 134 ESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDT Articulation- LMISRTPEVTCVVVDVSQEDPEVQFNWYVDG CH2-CH3 VEVHNAKTKPREEQFNSTYLV

WLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTTPPVLDSDGSFFLYS RLTVDKSRWQEGNVFSCSVMHEALHNHYTQK SLSLSLGK

# SEQUENCES ANNOTATION 135 RWPESPKAQASSVPTAQPQAEGSLAKATTAP IgD-articulation- ATTRNTGRGGEEKKKEKEKEEQEERETKTPE Fc

CPSHTQPLGVYLLTPAVQDLWLRDKATFTCFV VGSDLKDAHLTWEVAGKVPTGGVEEGLLERH SNGSQSQHSRLTLPRSLWNAGTSVTCTLNHP SLPPQRLMALREPAAQAPVKLSLNLLASSDPP EAASWLLCEVSGFSPPNILLMWLEDQREVNTS GFAPARPPPQPGSTTFWAWSVLRVPAPPSPQ

PATYTCVVSHEDSRTLLNASRSLEVSYVTDH 136 FWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 153-179 of access number P10747) 137 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLF PGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWV CD28 (amino acids 114-179 of access number P10747) 138 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRS CD28 (amino acids 180-220 of P10747) 139 RSKRSRGGHSDYMNMTPRRPGPTRKHYQPY CD28 (GG LL to APPRDFAAYRS ) 140 KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRF 4-1BB PEEEEGGCEL (amino acids 214-255 from Q07011.1) 141 RVKFSRSADAPAYQQGQNQLYNELNLGRREE CD3 zeta

YDVLDKRRGRDPEMGGKPRRKNPQEGLYNEL QKDKMAEAYSEIGMKGERRRGKGHDGLYQGL STATKDTYDALHMQALPPR

ANNOTATION SEQUENCE # 142 CAGAACCCAGATCCCGCGGTATATCAACTGC GCGACTCAAAATCATCCGATAAGAGTGTCTG sequence of exon 1 TTTGTTTACTGACTTCGACAGTCAAACTAATG TCTCTCAGAGCAAAGATTCCGATGTCTACAT constant region CACTGACAAGTGC recombinant alpha partial CRT YMLDLQPET 143 HPV 16 E7 peptide (11- 19) 144 gRNA of NNNNNNNNNNNNNNNNNNNNGUUUUAGAGC

UAGAAAUAGCAAGUUAAAAUAAGGCUAGUC TRAC CGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGC 145 NNNNNNNNNNNNNNNNNNNNGUUUUAGAGC GRNA

UAUGCUGAAAAGCAUAGCAAGUUAAAAUAA TRAC GGCUAGUCCGUUAUCAACUUGAAAAAGUGG

CACCGAGUCGGUGC 146 NNNNNNNNNNNNNNNNNNNNGUUUUAGAGC GRNA from

UAUGCUGGAAACAGCAUAGCAAGUUAAAAU TRAC AAGGCUAGUCCGUUAUCAACUUGAAAAAGU

GGCACCGAGUCGGUGC 147 NNNNNNNNNNNNNNNNNNNNGGUUUUAGAGC GRNA from

UAUGCUGUUUUGGAAACAAAACAGCAUAGC TRAC AAGUUAAAAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGC 148 NNNNNNNNNNNNNNNNNNNNGUAUUAGAGC GRNA from

UAGAAAUAGCAAGUUAAUAUAAGGCUAGUC TRAC CGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGC 149 NNNNNNNNNNNNNNNNNNNNGUUUAAGAGC GRNA

UAGAAAUAGCAAGUUUAAAUAAGGCUAGUC TRAC CGUUAUCAACUUGAAAAAGUGGCACCGAGU

CGGUGC 150 NNNNNNNNNNNNNNNNNNNNGUAUUAGAGC GRNA

# SEQUENCES NOTE UAUGCUGUAUUGGAAACAAUACAGCAUAGC TRAC AAGUUAAUAUAAGGCUAGUCCGUUAUCAAC

UUGAAAAAGUGGCACCGAGUCGGUGC 151 AAGGCUAGUCCGUUAUCAACUUGAAAAAGU proximal GGCACCGAGUCGGUGCU domain and gRNA tail 152 AAGGCUAGUCCGUUAUCAACUUGAAAAAGU proximal GGCACCGAGUCGGUGGUGC domain and tail gRNA 153 AAGGCUAGUCCGUUAUCAACUUGAAAAAGU proximal GGCACCGAGUCGGUGCGGAUC domain and tail gRNA 154 AAGGCUAGUCCGUUAUCAACUUGAAAAAGU proximal G domain and tail gRNA 155 AAGGCUAGUCCGUUAUCA proximal domain and gRNA tail 156 AAGGCUAGUCCG proximal and tail gRNA domain 157 NNNNNNNNNNNNNNNNNNNNGUUUUAGAGC GRNA UAGAAAUAGCAAGUUAAAAUAAGGCUAGUC chimeric CGUUAUCAACUUGAAAAGAGGGACACGAGAR

CGGUGCUUUUUU 158 NNNNNNNNNNNNNNNNNNNNGUUUUAGUAC GRNA UCUGGAAACAGAAUCUACUAAAACAAGGCA Chimeric AAAUGCCGUGUUUAUCUCGUCAACUUGUUG exemplary

GCGAGAUUUUUU 159 KKPYSIGLDIGTNSVGWAVVTDDYKVPAKKMK Streptococcus VLGNTDKSHIEKNLLGALLFDSGNTAEDRRLKR mutans Cas9

TARRRYTRRRNRILYLQEIFSEEMGKVDDSFFH RLEDSFLVTEDKRGERHPIFGNLEEEVKYHEN FPTIYHLRQYLADNPEKVDLRLVYLALAHIIKFR GHFLIEGKFDTRNNDVQRLFQEFLAVYDNTFE # SEQUENCES NOTE NSSLQEQNVQVEEILTDKISKSAKKDRVLKLFP NEKSNGRFAEFLKLIVGNQADFKKHFELEEKAP LQFSKDTYEEELEVLLAQIGDNYAELFLSAKKL YDSILLSGILTVTDVGTKAPLSASMIQRYNEHQ MDLAQLKQFIRQKLSDKYNEVFSDVSKDGYAG YIDGKTNQEAFYKYLKGLLNKIEGSGYFLDKIER EDFLRKQRTFDNGSIPHQIHLQEMRAIIRRQAE FYPFLADNQDRIEKLLTFRIPYYVGPLARGKSD FAWLSRKSADKITPWNFDEIVDKESSAEAFINR MTNYDLYLPNQKVLPKHSLLYEKFTVYNELTKV KYKTEQGKTAFFDANMKQEIFDGVFKVYRKVT KDKLMDFLEKEFDEFRIVDLTGLDKENKVFNAS YGTYHDLCKILDKDFLDNSKNEKILEDIVLTLTLF EDREMIRKRLENYSDLLTKEQVKKLERRHYTG WGRLSAELIHGIRNKESRKTILDYLIDDGNSNR NFMQLINDDALSFKEEIAKAQVIGETDNLNQVV SDIAGSPAIKKGILQSLKIVDELVKIMGHQPENIV VEMARENQFTNQGRRNSQQRLKGLTDSIKEF GSQILKEHPVENSQLQNDRLFLYYLQNGRDMY TGEELDIDYLSQYDIDHIIPQAFIKDNSIDNRVLT SSKENRGKSDDVPSKDVVRKMKSYWSKLLSA KLITQRKFDNLTKAERGGLTDDDKAGFIKRQLV ETRQITKHVARILDERFNTETDENNKKIRQVKIV TLKSNLVSNFRKEFELYKVREINDYHHAHDAYL NAVIGKALLGVYPQLEPEFVYGDYPHFHGHKE NKATAKKFFYSNIMNFFKKDDVRTDKNGEIIWK KDEHISNIKKVLSYPQVNIVKKVEEQTGGFSKE SILPKGNSDKLIPRKTKKFYWDTKKYGGFDSPI VAYSILVIADIEKGKSKKLKTVKALVGVTIMEKM TFERDPVAFLERKGYRNVQEENIIKLPKYSLFK LENGRKRLLASARELQKGNEIVLPNHLGTLLYH AKNIHKVDEPKHLDYVDKHKDEFKELLDVVSNF SKKYTLAEGNLEKIKELYAQNNGEDLKELASSF INLLTFTAIGAPATFKFFDKNIDRKRYTSTTEILN

ATLIHQSITGLYETRIDLNKLGGD 160 DKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKV Streptococcus

# SEQUENCES NOTES LGNTDRHSIKKNLIGALLFDSGETAEATRLKRT pyogenes Cas9

ARRRYTRRKNRICYLQEIFSNEMAKVDDSFFH RLEESFLVEEDKKHERHPIFGNIVDEVAYHEKY PTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEEN PINASGVDAKAILSARLSKSRRLENLIAQLPGEK KNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTL LKALVRQQLPEKYKEIFFDQSKNGYAGYIDGG ASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFL KDNREKIEKILTFRIPYYVGPLARGNSRFAWMT RKSEETITPWNFEEVVDKGASAQSFIERMTNF DKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQ LKEDYFKKIECFDSVEISGVEDRFNASLGTYHD LLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMI EERLKTYAHLFDDKVMKQLKRRRYTGWGRLS RKLINGIRDKQSGKTILDFLKSDGFANRNFMQLI HDDSLTFKEDIQKAQVSGQGDSLHEHIANLAG SPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQI LKEHPVENTQLQNEKLYLYYLQNGRDMYVDQ ELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRS DKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLI TQRKFDNLTKAERGGLSELDKAGFIKRQLVET RQITKHVAQILDSRMNTKYDENDKLIREVKVITL KSKLVSDFRKDFQFYKVREINNYHHAHDAYLN AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIA KSEQEIGKATAKYFFYSNIMNFFKTEITLANGEI RKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDN # SEQUENCES NOTE EQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG

LYETRIDLSQLGGD 161 TKPYSIGLDIGTNSVGWAVTTDNYKVPSKKMK Streptococcus VLGNTSKKYIKKNLLGVLLFDSGITAEGRRLKR thermophilus TARRRYTRRRNRILYLQEIFSTEMATLDDAFFQ Cas9

RLDDSFLVPDDKRDSKYPIFGNLVEEKAYHDE FPTIYHLRKYLADSTKKADLRLVYLALAHMIKYR GHFLIEGEFNSKNNDIQKNFQDFLDTYNAIFES DLSLENSKQLEEIVKDKISKLEKKDRILKLFPGE KNSGIFSEFLKLIVGNQADFRKCFNLDEKASLH FSKESYDEDLETLLGYIGDDYSDVFLKAKKLYD AILLSGFLTVTDNETEAPLSSAMIKRYNEHKEDL ALLKEYIRNISLKTYNEVFKDDTKNGYAGYIDGK TNQEDFYVYLKKLLAEFEGADYFLEKIDREDFL RKQRTFDNGSIPYQIHLQEMRAILDKQAKFYPF LAKNKERIEKILTFRIPYYVGPLARGNSDFAWSI RKRNEKITPWNFEDVIDKESSAEAFINRMTSFD LYLPEEKVLPKHSLLYETFNVYNELTKVRFIAES MRDYQFLDSKQKKDIVRLYFKDKRKVTDKDIIE YLHAIYGYDGIELKGIEKQFNSSLSTYHDLLNIIN DKEFLDDSSNEAIIEEIIHTLTIFEDREMIKQRLS KFENIFDKSVLKKLSRRHYTGWGKLSAKLINGI RDEKSGNTILDYLIDDGISNRNFMQLIHDDALSF KKKIQKAQIIGDEDKGNIKEVVKSLPGSPAIKKGI LQSIKIVDELVKVMGGRKPESIVVEMARENQYT NQGKSNSQQRLKRLEKSLKELGSKILKENIPAK LSKIDNNALQNDRLYLYYLQNGKDMYTGDDLDI DRLSNYDIDHIIPQAFLKDNSIDNKVLVSSASNR GKSDDVPSLEVVKKRKTFWYQLLKSKLISQRK FDNLTKAERGGLSPEDKAGFIQRQLVETRQITK HVARLLDEKFNNKKDENNRAVRTVKIITLKSTL VSQFRKDFELYKVREINDFHHAHDAYLNAVVA SALLKKYPKLEPEFVYGDYPKYNSFRERKSAT EKVYFYSNIMNIFKKSISLADGRVIERPLIEVNEE # SEQUENCES NOTE TGESVWNKESDLATVRRVLSYPQVNVVKKVE EQNHGLDRGKPKGLFNANLSSKPKPNSNENL VGAKEYLDPKKYGGYAGISNSFTVLVKGTIEKG AKKKITNVLEFQGISILDRINYRKDKLNFLLEKG YKDIELIIELPKYSLFELSDGSRRMLASILSTNNK RGEIHKGNQIFLSQKFVKLLYHAKRISNTINENH RKYVENHKKEFEELFYYILEFNENYVGAKKNG KLLNSAFQSWQNHSIDELCSSFIGPTGSERKG LFELTSRGSAADFEFLGVKIPRYRDYTPSSLLK

DATLIHQSVTGLYETRIDLAKLGEG 162 KKPYTIGLDIGTNSVGWAVLTDQYDLVKRKMKI Listeria innocua AGDSEKKQIKKNFWGVRLFDEGQTAADRRMA Cas9

RTARRRIERRRNRISYLQGIFAEEMSKTDANFF CRLSDSFYVDNEKRNSRHPFFATIEEEVEYHK NYPTIYHLREELVNSSEKADLRLVYLALAHIIKY RGNFLIEGALDTQNTSVDGIYKQFIQTYNQVFA SGIEDGSLKKLEDNKDVAKILVEKVTRKEKLERI LKLYPGEKSAGMFAQFISLIVGSKGNFQKPFDL IEKSDIECAKDSYEEDLESLLALIGDEYAELFVA AKNAYSAVVLSSIITVAETETNAKLSASMIERFD THEEDLGELKAFIKLHLPKHYEEIFSNTEKHGY AGYIDGKTKQADFYKYMKMTLENIEGADYFIAK IEKENFLRKQRTFDNGAIPHQLHLEELEAILHQ QAKYYPFLKENYDKIKSLVTFRIPYFVGPLANG QSEFAWLTRKADGEIRPWNIEEKVDFGKSAVD FIEKMTNKDTYLPKENVLPKHSLCYQKYLVYNE LTKVRYINDQGKTSYFSGQEKEQIFNDLFKQKR KVKKKDLELFLRNMSHVESPTIEGLEDSFNSSY STYHDLLKVGIKQEILDNPVNTEMLENIVKILTVF EDKRMIKEQLQQFSDVLDGVVLKKLERRHYTG WGRLSAKLLMGIRDKQSHLTILDYLMNDDGLN RNLMQLINDSNLSFKSIIEKEQVTTADKDIQSIV ADLAGSPAIKKGILQSLKIVDELVSVMGYPPQTI VVEMARENQTTGKGKNNSRPRYKSLEKAIKEF GSQILKEHPTDNQELRNNRLYLYYLQNGKDMY TGQDLDIHNLSNYDIDHIVPQSFITDNSIDNLVLT # SEQUENCES NOTE SSAGNREKGDDVPPLEIVRKRKVFWEKLYQG NLMSKRKFDYLTKAERGGLTEADKARFIHRQL VETRQITKNVANILHQRFNYEKDDHGNTMKQV RIVTLKSALVSQFRKQFQLYKVRDVNDYHHAH DAYLNGVVANTLLKVYPQLEPEFVYGDYHQFD WFKANKATAKKQFYTNIMLFFAQKDRIIDENGE ILWDKKYLDTVKKVMSYRQMNIVKKTEIQKGEF SKATIKPKGNSSKLIPRKTNWDPMKYGGLDSP NMAYAVVIEYAKGKNKLVFEKKIIRVTIMERKAF EKDEKAFLEEQGYRQPKVLAKLPKYTLYECEE GRRRMLASANEAQKGNQQVLPNHLVTLLHHA ANCEVSDGKSLDYIESNREMFAELLAHVSEFA KRYTLAEANLNKINQLFEQNKEGDIKAIAQSFV DLMAFNAMGAPASFKFFETTIERKRYNNLKELL

NSTIIYQSITGLYESRKRLDD 163 GAGAATCAAAATCGGTGAAT Targeting sequence 2

TRAC 164 GGCCTCGGCGCTGACGATCT Targeting sequence 2

TRBC 165 GAGAATCAAAATCGGTGAATAGG TRAC targeting sequence with PAM 3 166 TTCAAAACCTGTCAGTGATTGGG TRAC targeting sequence with PAM 4 167 TGTGCTAGACATGAGGTCTATGG TRAC targeting sequence with PAM 5 168 CGTCATGAGCAGATTA

# SEQUENCES ANNOTATION PAM 6 169 TCAGGGTTCTGGATATCTGTGGG TRAC targeting sequence with PAM 7 170 GTCAGGGTTCTGGATATCTGTGG TRAC targeting sequence with PAM 8 171 TTCGGAACCCAATCACTGACAGG Targeting sequence TRAC with PTCCCAGGAGGAGGAGGGG 174 AACAAATGTGTCACAAAGTAAGG TRAC targeting sequence with PAM 12 175 TGGATTTAGAGTCTCTCAGCTGG TRAC targeting sequence with PAM 13 176 TAGGCAGACAGACTTGTCACTGG TRAC targeting sequence with PAM 14 177 AGCTGGTACACGGCAGGGAGTCAGGAGAGGAGAGTCTCGGAGAGAGGTCTCGGAGAGGAGTCTCGG

# SEQUENCE ANNOTATION TRAC with MBP 15,178 GCTGGTACACGGCAGGGTCAGGG TRAC targeting sequence with MBP 16,179 TCTCTCAGCTGGTACACGGCAGG TRAC targeting sequence with MBP 17,180 TTTCAAAACCTGTCAGTGATTGG TRAC targeting sequence with MBP 18,181 GATTAAACCCGGCCACTTTCAGG TRAC targeting sequence with MBP 19,182 CTCGACCAGCTTGACATCACAGG TRAC targeting Sequence PAM 20 183 AGAGTCTCTCAGCTGGTACACGG TRAC targeting sequence with PAM 21 184 CTCTCAGCTGGTACACGGCAGGG TRAC targeting sequence with PAM 22 185 AAGTTCCTGTGATGTCAAGCTGG TRAC targeting sequence with PAM 23 186 ATCCTCCTCCTCCTAGTGA

# SEQUENCE ANNOTATION TRAC direction with MBP 24,187 TGCTCATGACGCTGCGGCTGTGG TRAC targeting sequence with MBP 25,188 ACAAAACTGTGCTAGACATGAGG TRAC targeting sequence with MBP 26,189 ATTTGTTTGAGAATCAAAATCGG TRAC targeting sequence with MBP 27,190 CATCACAGGAACTTTCTAAAAGG TRAC targeting sequence with MBP 28,191 GTCGAGAAAAGCTTTGAAACAGG targeting sequence TRAC with PAM 29 192 CCACTTTCAGGAGGAGGATTCGG TRAC targeting sequence with PAM 30 193 CTGACAGGTTTTGAAAGTTTAGG TRAC targeting sequence with PAM 31 194 AGCTTTGAAACAGGTAAGACAGG TRAC targeting sequence with PAM 32

# SEQUENCE LEC 195 TGGAATAATGCTGTTGTTGAAGG TRAC targeting sequence with MBP 33,196 AGAGCAACAGTGCTGTGGCCTGG TRAC targeting sequence with MBP 34,197 CTGTGGTCCAGCTGAGGTGAGGG TRAC targeting sequence with MBP 35,198 CTGCGGCTGTGGTCCAGCTGAGG TRAC targeting sequence with MBP 36,199 TGTGGTCCAGCTGAGGTGAGGGG TRAC targeting sequence with MBP 37,200 CTTCTTCCCCAGCCCAGGTAAGG TRAC targeting sequence with PAM 38 201 ACACGGCAGGGTCAGGGTTCTGG TRAC targeting sequence with PAM 39 202 CTTCAAGAGCAACAGTGCTGTGG TRAC targeting sequence with PAM 40 203 CTGGGGAAGAAGGTGTCTTCTGG TRAC targeting sequence with

# SEQUENCE ANNOTATION PAM 41 204 TCCTCCTCCTGAAAGTGGCCGGG TRAC targeting sequence with MBP 42,205 TTAATCTGCTCATGACGCTGCGG TRAC targeting sequence with MBP 43,206 ACCCGGCCACTTTCAGGAGGAGG TRAC targeting sequence with PAM 44 207 TTCTTCCCCAGCCCAGGTAAGGG TRAC targeting sequence with MBP 45,208 CTTACCTGGGCTGGGGAAGAAGG TRAC targeting sequence with PAM 46 209 GACACCTTCTTCCCCAGCCCAGG TRAC target sequence with PAM 47 210 GCTGTGGTCCAGCTGAGGTGAGG TRAC target sequence with PAM 48 211 CCGAATCCTCCTCCTGAAAGTGG TRAC target sequence with PAM 49 212 GCTGTCAAGTCCAGT

# SEQUENCES ANNOTATION TRBC with PAM 3 213 CTATGGACTTCAAGAGCAACAGTGCTGT Targeting sequence 1

TRAC ZFN 214 CTCATGTCTAGCACAGTTTTGTCTGTGA Targeting sequence 2

TRAC ZFN 215 GTGCTGTGGCCTGGAGCAACAAATCTGA Targeting sequence 3

TRAC ZFN 216 TTGCTCTTGAAGTCCATAGACCTCATGT Targeting sequence 4

TRAC ZFN 217 GCTGTGGCCTGGAGCAACAAATCTGACT Targeting sequence 5

TRAC ZFN 218 CTGTTGCTCTTGAAGTCCATAGACCTCA Targeting sequence 6

TRAC ZFN 219 CTGTGGCCTGGAGCAACAAATCTGACTT Targeting sequence 7

TRAC ZFN 220 CTGACTTTGCATGTGCAAACGCCTTCAA Targeting sequence 8

TRAC ZFN 221 TTGTTGCTCCAGGCCACAGCACTGTTGC Targeting sequence 9

TRAC ZFN 222 TGAAAGTGGCCGGGTTTAATCTGCTCAT Targeting sequence 10

TRAC ZFN 223 AGGAGGATTCGGAACCCAATCACTGACA String 11 of

# SEQUENCES ANNOTATION targeting

TRAC ZFN 224 GAGGAGGATTCGGAACCCAATCACTGAC Targeting sequence 12

TRAC ZFN 225 CCGTAGAACTGGACTTGACAGCGGAAGT Targeting sequence 1

TRBC ZFN 226 TCTCGGAGAATGACGAGTGGACCCAGGA Targeting sequence 2

TRBC ZFN 227 MAAFKPNSINYILGLDIGIASVGWAMVEIDEEEN Neisseria PIRLIDLGVRVFERAEVPKTGDSLAMARRLARS meningitidis VRRLTRRRAHRLLRTRRLLKREGVLQAANFDE Cas9

NGLIKSLPNTPWQLRAAALDRKLTPLEWSAVLL HLIKHRGYLSQRKNEGETADKELGALLKGVAG NAHALQTGDFRTPAELALNKFEKESGHIRNQR SDYSHTFSRKDLQAELILLFEKQKEFGNPHVSG GLKEGIETLLMTQRPALSGDAVQKMLGHCTFE PAEPKAAKNTYTAERFIWLTKLNNLRILEQGSE RPLTDTERATLMDEPYRKSKLTYAQARKLLGL EDTAFFKGLRYGKDNAEASTLMEMKAYHAISR ALEKEGLKDKKSPLNLSPELQDEIGTAFSLFKT DEDITGRLKDRIQPEILEALLKHISFDKFVQISLK ALRRIVPLMEQGKRYDEACAEIYGDHYGKKNT EEKIYLPPIPADEIRNPVVLRALSQARKVINGVV RRYGSPARIHIETAREVGKSFKDRKEIEKRQEE NRKDREKAAAKFREYFPNFVGEPKSKDILKLRL YEQQHGKCLYSGKEINLGRLNEKGYVEIDHAL PFSRTWDDSFNNKVLVLGSENQNKGNQTPYE YFNGKDNSREWQEFKARVETSRFPRSKKQRIL LQKFDEDGFKERNLNDTRYVNRFLCQFVADR MRLTGKGKKRVFASNGQITNLLRGFWGLRKV RAENDRHHALDAVVVACSTVAMQQKITRFVRY KEMNAFDGKTIDKETGEVLHQKTHFPQPWEFF # SEQUENCES NOTE AQEVMIRVFGKPDGKPEFEEADTLEKLRTLLAE KLSSRPEAVHEYVTPLFVSRAPNRKMSGQGH METVKSAKRLDEGVSVLRVPLTQLKLKDLEKM VNREREPKLYEALKARLEAHKDDPAKAFAEPF YKYDKAGNRTQQVKAVRVEQVQKTGVWVRN HNGIADNATMVRVDVFEKGDKYYLVPIYSWQV AKGILPDRAVVQGKDEEDWQLIDDSFNFKFSL HPNDLVEVITKKARMFGYFASCHRGTGNINIRI HDLDHKIGKNGILEGIGVKTALSFQKYQIDELGK

EIRPCRLKKRPPVR 228 MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFK Streptococcus VLGNTDRHSIKKNLIGALLFDSGETAEATRLKR pyogenes Cas9

TARRRYTRRKNRICYLQEIFSNEMAKVDDSFF HRLEESFLVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFR GHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSD AILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDG GASQEEFYKFIKPILEKMDGTEELLVKLNREDL LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYP FLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMT NFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVK QLKEDYFKKIECFDSVEISGVEDRFNASLGTYH DLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDRE MIEERLKTYAHLFDDKVMKQLKRRRYTGWGRL SRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLA GSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE MARENQTTQKGQKNSRERMKRIEEGIKELGS QILKEHPVENTQLQNEKLYLYYLQNGRDMYVD QELDINRLSDYDVDHIVPQSFLKDDSIDNKVLT # SEQUENCES NOTE RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNA KLITQRKFDNLTKAERGGLSELDKAGFIKRQLV ETRQITKHVAQILDSRMNTKYDENDKLIREVKVI TLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMI AKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSM PQVNIVKKTEVQTGGFSKESILPKRNSDKLIAR KKDWDPKKYGGFDSPTVAYSVLVVAKVEKGK SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGY KEVKKDLIIKLPKYSLFELENGRKRMLASAGEL QKGNELALPSKYVNFLYLASHYEKLKGSPEDN EQKQLFVEQHKHYLDEIIEQISEFSKRVILADAN LDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGA PAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITG

LYETRIDLSQLGGD 229 AGCTGAGAGACTCTAAATCCAGTGACAAGTC TRAC 50 bp TGTCTGCCTATTCACCGAT Homology arm 5 '230 CTGATCCTCTTGTCCCACAGATATCCAGAAC Arm of 5 ’CCTGACCCTGCCGTGTACCAGCTGTACT

CACCGAT TRAC 231 GTGACTTGCCAGCCCCACAGAGCCCCGCCC 5 'arm TTGTCCATCACTGGCATCTGGACTCCAGCCT Homology of GGGTTGGGGCAAAGAGGGAAATGAGATCAT 200 bp of

GTCCTAACCCTGATCCTCTTGTCCCACAGAT TRAC ATCCAGAACCCTGACCCTGCCGTGTACCAG CTGAGAGACTCTAAATCCAGTGACAAGTCTG

TCTGCCTATTCACCGAT 232 CCTCTTGGCCAAGATTGATAGCTTGTGCCTG 5 'arm TCCCTGAGTCCCAGTCCATCACGAGCAGCT Homology of GGTTTCTAAGATGCTATTTCCCGTATAAAGCA 300 bp from

TGAGACCGTGACTTGCCAGCCCCACAGAGC TRAC CCCGCCCTTGTCCATCACTGGCATCTGGACT CCAGCCTGGGTTGGGGCAAAGAGGGAAATG # SEQUENCES NOTE AGATCATGTCCTAACCCTGATCCTCTTGTCC CACAGATATCCAGAACCCTGACCCTGCCGTG TACCAGCTGAGAGACTCTAAATCCAGTGACA

AGTCTGTCTGCCTATTCACCGAT 233 ATTAAATAAAAGAATAAGCAGTATTATTAAGT 5 'arm AGCCCTGCATTTCAGGTTTCCTTGAGTGGCA GGCCAGGCCTGGCCGTGAACGTTCACTGAA 400 bp homology

ATCATGGCCTCTTGGCCAAGATTGATAGCTT TRAC GTGCCTGTCCCTGAGTCCCAGTCCATCACGA GCAGCTGGTTTCTAAGATGCTATTTCCCGTA TAAAGCATGAGACCGTGACTTGCCAGCCCCA CAGAGCCCCGCCCTTGTCCATCACTGGCAT CTGGACTCCAGCCTGGGTTGGGGCAAAGAG GGAAATGAGATCATGTCCTAACCCTGATCCT CTTGTCCCACAGATATCCAGAACCCTGACCC TGCCGTGTACCAGCTGAGAGACTCTAAATCC

AGTGACAAGTCTGTCTGCCTATTCACCGAT 234 CTCCAGATTCCAAGATGTACAGTTTGCTTTG 5 'Arm CTGGGCCTTTTTCCCCATGCCTGCCTTTACTC TGCCAGAGTTATATTGCTGGGGTTTTGAAGA 500 b

AGATCCTATTAAATAAAAGAATAAGCAGTATT TRAC ATTAAGTAGCCCTGCATTTCAGGTTTCCTTGA GTGGCAGGCCAGGCCTGGCCGTGAACGTTC ACTGAAATCATGGCCTCTTGGCCAAGATTGA TAGCTTGTGCCTGTCCCTGAGTCCCAGTCCA TCACGAGCAGCTGGTTTCTAAGATGCTATTT CCCGTATAAAGCATGAGACCGTGACTTGCCA GCCCCACAGAGCCCCGCCCTTGTCCATCAC TGGCATCTGGACTCCAGCCTGGGTTGGGGC AAAGAGGGAAATGAGATCATGTCCTAACCCT GATCCTCTTGTCCCACAGATATCCAGAACCC TGACCCTGCCGTGTACCAGCTGAGAGACTCT AAATCCAGTGACAAGTCTGTCTGCCTATTCA

CCGAT 235 AGAGAGCAATCTCCTGGTAATGTGATAGATT 5 'TCCCAACTTAATGCCAACATACCATAAACCTC Arm Homology of

# SEQUENCES ANNOTATION CCATTCTGCTAATGCCCAGCCTAAGTTGGGG 600 bp of

AGACCACTCCAGATTCCAAGATGTACAGTTT TRAC GCTTTGCTGGGCCTTTTTCCCATGCCTGCCT TTACTCTGCCAGAGTTATATTGCTGGGGTTTT GAAGAAGATCCTATTAAATAAAAGAATAAGCA GTATTATTAAGTAGCCCTGCATTTCAGGTTTC CTTGAGTGGCAGGCCAGGCCTGGCCGTGAA CGTTCACTGAAATCATGGCCTCTTGGCCAAG ATTGATAGCTTGTGCCTGTCCCTGAGTCCCA GTCCATCACGAGCAGCTGGTTTCTAAGATGC TATTTCCCGTATAAAGCATGAGACCGTGACT TGCCAGCCCCACAGAGCCCCGCCCTTGTCC ATCACTGGCATCTGGACTCCAGCCTGGGTTG GGGCAAAGAGGGAAATGAGATCATGTCCTAA CCCTGATCCTCTTGTCCCACAGATATCCAGA ACCCTGACCCTGCCGTGTACCAGCTGAGAG ACTCTAAATCCAGTGACAAGTCTGTCTGCCT

ATTCACCGAT 236 TTTGATTCTCAAACAAATGTGTCACAAAGTAA TRAC 50 bp 3 'GGATTCTGATGTGTATAT Homology Arm 237 TTTGATTCTCAAACAAATGTGTCACAAAGTAA de 3 ’GGATTCTGATGTAGATT

GCAACA TRAC 238 TTTGATTCTCAAACAAATGTGTCACAAAGTAA 3 'arm GGATTCTGATGTGTATATCACAGACAAAACT GTGCTAGACATGAGGTCTATGGACTTCAAGA 200 bp homology

GCAACAGTGCTGTGGCCTGGAGCAACAAAT TRAC CTGACTTTGCATGTGCAAACGCCTTCAACAA CAGCATTATTCCAGAAGACACCTTCTTCCCC

AGCCCAGGTAAGGG 239 TTTGATTCTCAAACAAATGTGTCACAAAGTAA 3 'Arm GGATTCTGATGTGTATATCACAGACAAAACT Homology of GTGCTAGACATGAGGTCTATGGACTTCAAGA 300 bp from

GCAACAGTGCTGTGGCCTGGAGCAACAAAT TRAC CTGACTTTGCATGTGCAAACGCCTTCAACAA # SEQUENCES NOTE CAGCATTATTCCAGAAGACACCTTCTTCCCC AGCCCAGGTAAGGGCAGCTTTGGTGCCTTC GCAGGCTGTTTCCTTGCTTCAGGAATGGCCA GGTTCTGCCCAGAGCTCTGGTCAATGATGTC

TAAAACTCCTCTGATTGGTGGT 240 TTTGATTCTCAAACAAATGTGTCACAAAGTAA 3 'Arm GGATTCTGATGTGTATATCACAGACAAAACT GTGCTAGACATGAGGTCTATGGACTTCAAGA 400 bp homology

GCAACAGTGCTGTGGCCTGGAGCAACAAAT TRAC CTGACTTTGCATGTGCAAACGCCTTCAACAA CAGCATTATTCCAGAAGACACCTTCTTCCCC AGCCCAGGTAAGGGCAGCTTTGGTGCCTTC GCAGGCTGTTTCCTTGCTTCAGGAATGGCCA GGTTCTGCCCAGAGCTCTGGTCAATGATGTC TAAAACTCCTCTGATTGGTGGTCTCGGCCTT ATCCATTGCCACCAAAACCCTCTTTTTACTAA GAAACAGTGAGCCTTGTTCTGGCAGTCCAGA

GAATGACACGGGAAAAAAGCAGATGAAG 241 TTTGATTCTCAAACAAATGTGTCACAAAGTAA 3 'arm GGATTCTGATGTGTATATCACAGACAAAACT GTGCTAGACATGAGGTCTATGGACTTCAAGA 500 bp homology

GCAACAGTGCTGTGGCCTGGAGCAACAAAT TRAC CTGACTTTGCATGTGCAAACGCCTTCAACAA CAGCATTATTCCAGAAGACACCTTCTTCCCC AGCCCAGGTAAGGGCAGCTTTGGTGCCTTC GCAGGCTGTTTCCTTGCTTCAGGAATGGCCA GGTTCTGCCCAGAGCTCTGGTCAATGATGTC TAAAACTCCTCTGATTGGTGGTCTCGGCCTT ATCCATTGCCACCAAAACCCTCTTTTTACTAA GAAACAGTGAGCCTTGTTCTGGCAGTCCAGA GAATGACACGGGAAAAAAGCAGATGAAGAG AAGGTGGCAGGAGAGGGCACGTGGCCCAG CCTCAGTCTCTCCAACTGAGTTCCTGCCTGC CTGCCTTTGCTCAGACTGTTTGCCCCTTACT

GCTCTTC 242 TTTGATTCTCAAACAAATGTGTCACAAAGTAA 3 'arm

# SEQUENCES ANNOTATION GGATTCTGATGTGTATATCACAGACAAAACT Homology of GTGCTAGACATGAGGTCTATGGACTTCAAGA 600 bp

GCAACAGTGCTGTGGCCTGGAGCAACAAAT TRAC CTGACTTTGCATGTGCAAACGCCTTCAACAA CAGCATTATTCCAGAAGACACCTTCTTCCCC AGCCCAGGTAAGGGCAGCTTTGGTGCCTTC GCAGGCTGTTTCCTTGCTTCAGGAATGGCCA GGTTCTGCCCAGAGCTCTGGTCAATGATGTC TAAAACTCCTCTGATTGGTGGTCTCGGCCTT ATCCATTGCCACCAAAACCCTCTTTTTACTAA GAAACAGTGAGCCTTGTTCTGGCAGTCCAGA GAATGACACGGGAAAAAAGCAGATGAAGAG AAGGTGGCAGGAGAGGGCACGTGGCCCAG CCTCAGTCTCTCCAACTGAGTTCCTGCCTGC CTGCCTTTGCTCAGACTGTTTGCCCCTTACT GCTCTTCTAGGCCTCATTCTAAGCCCCTTCT CCAAGTTGCCTCTCCTTATTTCTCCCTGTCTG CCAAAAAATCTTTCCCAGCTCACTAAGTCAG

TCTCACGCAGTCA 243 NIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA of human TCR WSNKSDFACANAFNNSIIPADTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 244 HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA of TCR WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 245 YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA of TCR WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 246 DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKTVLDMRSMDFKSNSAVA of TCR

# SEQUENCES NOTES human WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 247 PNIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQ Alpha Constant TNVSQSKDSDVYITDKTVLDMRSMDFKSNSAV of TCR AWSNKSDFACANAFNNSIIPADTFFPSPESSCD

VKLVEKSFETDTNLNFQNLSVIGFRILLLKVAGF

NLLMTLRLWSS 248 NIQKPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA of TCR WSNKSDFACANAFNNSIIPADTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 249 HIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA TCR WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 250 YIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA of human TCR WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 251 NIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA of human TCR WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 252 DIQNPDPAVYQLRDSKSSDKSVCLFTDFDSQT Alpha constant NVSQSKDSDVYITDKCVLDMRSMDFKSNSAVA of human TCR WSNKSDFACANAFNNSIIPEDTFFPSPESSCDV

KLVEKSFETDTNLNFQNLSVIGFRILLLKVAGFN

LLMTLRLWSS 253 EDLNKVFPPEVAVFEPSEAEISHTQKATLVCLA Beta constant TGFFPDHVELSWWVNGKEVHSGVCTDPQPLK of TCR human EQPALNDSRYCLSSRLRVSATFWQNPRNHFR

# SEQUENCES NOTE CQVQFYGLSENDEWTQDRAKPVTQIVSAEAW GRADCGFTSVSYQQGVLSATILYEILLGKATLY

AVLVSALVLMAMVKRKDF 254 TEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL Beta constant ATGFYPDHVELSWWVNGKEVHSGVSTDPQPL of human TCR KEQPALNDSRYCLSSRLRVSATFWQNPRNHF

RCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATL

YAVLVSALVLMAMVKRKDSRG 255 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL Beta constant ATGFYPDHVELSWWVNGKEVHSGVSTDPQPL of human TCR KEQPALNDSRYCLSSRLRVSATFWQNPRNHF

RCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATL

YAVLVSALVLMAMVKRKDSRG 256 EDLKNVFPPEVAVFEPSEAEISHTQKATLVCLA Beta constant TGFYPDHVELSWWVNGKEVHSGVCTDPQPLK of TCR EQPALNDSRYCLSSRLRVSATFWQNPRNHFR

CQVQFYGLSENDEWTQDRAKPVTQIVSAEAW GRADCGFTSESYQQGVLSATILYEILLGKATLY

AVLVSALVLMAMVKRKDSRG 257 TEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL Beta constant ATGFYPDHVELSWWVNGKEVHSGVCTDPQPL of human TCR KEQPALNDSRYCLSSRLRVSATFWQNPRNHF

RCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATL

YAVLVSALVLMAMVKRKDSRG 258 LEDLKNVFPPEVAVFEPSEAEISHTQKATLVCL Beta constant ATGFYPDHVELSWWVNGKEVHSGVCTDPQPL of human TCR KEQPALNDSRYCLSSRLRVSATFWQNPRNHF

RCQVQFYGLSENDEWTQDRAKPVTQIVSAEA WGRADCGFTSESYQQGVLSATILYEILLGKATL

YAVLVSALVLMAMVKRKDSRG 259 QNPDPAVYQLRDSKSSDKSVCLFTDFDSQTNV Region SQSKDSDVYITDKC partial TCR alpha constant

# SEQUENCES ANNOTATION 260 GTGCTAGACATGAGGTCTATGGACTTCAAGA Region GCAACAGTGCTGTGGCCTGGAGCAACAAAT alpha constant CTGACTTTGCATGTGCAAACGCCTTCAACAA part of TCR CAGCATTATTCCAGAAGACCTGTACHTTTTTTTTTTTTTTTF

Claims (210)

1. Genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, said modified TRAC locus that comprises a nucleic acid encoding a recombinant TCR or part of it, the said recombinant TCR or part thereof comprising: (i) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), and (ii) a beta TCR chain (TCRβ) which comprises a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoded by TCRβ and the Vα domain, and (b) and an open reading frame the endogenous TRAC locus or a partial sequence thereof, where the open reading frame encodes at least part of the Cα domain of the recombinant TCR. 2. Genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, said modified TRAC locus that comprises a nucleic acid encoding a recombinant TCR or part of it, the said recombinant TCR or part thereof comprising: (i) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), and (ii) a beta TCR chain (TCRβ) which comprises a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoded by TCRβ and the Vα domain, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, in which the open reading frame encodes at least part of the recombinant TCR Cα domain, in which: a part of Cα is encoded by the open reading structure one of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, wherein said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, referred to one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more more non-native cysteines. 3. Genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, said modified TRAC locus which comprises a nucleic acid encoding a recombinant TCR or part of it, the said recombinant TCR or part thereof comprising: (i) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), and (ii) a beta TCR chain (TCRβ) which comprises a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoded by TCRβ and the Vα domain, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, where the open reading frame encodes at least part of the recombinant TCR Cα domain, where the transgene sequence comprises one or more heterologous or regulatory control elements comprising a he promoter a therapist, operably linked to control TCR expression when expressed from an introduced cell with the genetically modified T cell. 4. Genetically modified T cell according to any one of claims 1 to 3, characterized by the fact that the transgene sequence was integrated by means of homology-directed repair (HDR). 5. Genetically modified T cell according to any one of claims 1 to 4, characterized in that the modified TRAC locus comprises a fusion in structure of (i) a transgene sequence and (ii) and a reading structure open or a partial sequence of the same of the endogenous TRAC locus, optionally in which the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the endogenous TRAC locus. A genetically modified T cell according to any one of claims 1 to 5, characterized in that the transgene sequence does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron. 7. Genetically modified T cell according to any one of claims 1 and 3 to 6, characterized by the fact that a part of the Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and a another part of Cα is encoded by the transgene sequence, where said other part of Cα is smaller than the natural size of a native Cα. 8. Genetically modified T cell according to any one of claims 1 to 7, characterized by the fact that the open reading frame or the partial sequence of the same comprises at least one intron and at least one exon of the TRAC locus endogenous. 9. Genetically modified T cell according to any one of claims 2, 7 and 8, characterized by the fact that the other part of Cα is encoded by a nucleotide sequence comprising less than four exons, less than three exons, less than two exons, one exon, or less than one complete exon of the open reading structure of the TRAC locus. 10. Genetically modified T cell according to any of claims 2 and 7 to 9, characterized by the fact that the other part of Cα is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long. 11. Genetically modified T cell according to any of claims 2 and 7 to 10, characterized by the fact that the other part of Cα is encoded by an exon 1 part of the TRAC locus, in which the exon 1 part is smaller than the natural size of exon 1 of the reading frame opened from the TRAC locus. 12. Genetically modified T cell according to any one of claims 1 to 11, characterized by the fact that the transgene sequence has been integrated downstream of the more 5 'exon 1 nucleotide and upstream of the more 3' exon nucleotide 1 of the open reading structure of the endogenous TRAC locus. 13. T cell genetically modified according to claim 1 or 2, characterized by the fact that said at least a part of Cα is encoded by at least a part of exon 1 and exons 2 to 4 of the open reading structure of the locus Endogenous TRAC. 14. Genetically modified T cell according to any one of claims 1 to 13, characterized by the fact that the encoded TCRα chain is able to dimerize with a TCRβ chain. 15. Genetically modified T cell according to any one of claims 1 to 14, characterized by the fact that the encoded Cα comprises the selected sequence from any of SEQ ID NOS: 14, 15, 19, and 24, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity sequence with any of SEQ ID NOS: 14, 15, 19, and 24, or a partial sequence thereof. 16. Genetically modified T cell according to any one of claims 1 to 14, characterized by the fact that the encoded Cα comprises the selected sequence of any one of SEQ ID NOS: 19, 24 and 243 to 252, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity sequence with any of SEQ ID NOS: 19, 24 and 243 to 252, or a partial sequence of them. 17. Genetically modified T cell according to any of claims 2 and 7 to 16, characterized by the fact that said at least a part of Cα is encoded by a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of it, or a partial sequence of it. 18. Genetically modified T cell according to any of claims 2 and 7 to 17, characterized in that the other part of Cα comprises a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 142, or a partial sequence thereof. 19. Genetically modified T cell according to any one of claims 7 to 18, characterized in that the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, option - finally, a substitution, deletion, or insertion of one or more amino acids in comparison to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable to form one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more non-native cysteines. 20. Genetically modified T cell according to claim 2 or 19, characterized by the fact that the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. 21. Genetically modified T cell according to any of claims 2, 19 and 20, characterized by the fact that a coded Cα region comprises a cysteine in a position corresponding to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO: 20. 22. Genetically modified T cell according to any one of claims 2 and 19 to 21, characterized by the fact that the encoded Cα comprises the sequence selected from any one of SEQ ID NOS: 248 to 252, or a partial sequence from same. 23. Genetically modified T cell according to any of claims 1 to 22, characterized in that the modified T cell also comprises a genetic disruption at a TRBC locus. 24. Genetically modified T cell, characterized by the fact that it comprises a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus which comprises a nucleic acid encoding a recombinant TCR or part of it, said Recombinant TCR or part thereof comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the nucleic acid sequence comprises (a) a transgene sequence encoding TCRα and the Vβ domain, and (b) and a open reading frame of the endogenous TRBC locus or a partial sequence thereof, where the open reading frame encodes at least part of the Cβ domain of the recombinant TCR. 25. Genetically modified T cell, characterized by the fact that it comprises a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus which comprises a nucleic acid encoding a recombinant TCR or part of it, said Recombinant TCR or part thereof comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the nucleic acid sequence comprises (a) a transgene sequence encoding TCRα and the Vβ domain, and (b) and a open reading frame of the endogenous TRBC site or a partial sequence thereof, where the open reading frame encodes at least part of the domain Recombinant TCR Cβ, where: a part of Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of Cβ is encoded by the transgene sequence, where said other part of Cβ is smaller than the natural size of a native Cβ; and the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids compared to a region Native Cβ and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. 26. Genetically modified T cell according to claim 24 or 25, characterized by the fact that the transgene sequence has been integrated by means of homology-directed repair (HDR). 27. Genetically modified T cell according to any one of claims 24 to 26, characterized by the fact that the TRBC locus is a TRBC1 locus and / or a TRBC2 locus. 28. Genetically modified T cell according to any one of claims 24 to 27, characterized in that the modified TRBC locus comprises a structure fusion of (i) a transgene sequence and (ii) and an open reading frame or a partial sequence of the same of the endogenous TRBC locus, optionally in which the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the endogenous TRBC locus. 29. A genetically modified T cell according to any one of claims 24 to 28, characterized in that the transgene sequence does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron. 30. Genetically modified T cell according to any of claims 24 and 26 to 29, characterized in that a part of the Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of Cβ is encoded by the transgene sequence, where the said other part of Cβ is smaller than the natural size of a native Cβ. 31. Genetically modified T cell according to any one of claims 24 to 30, characterized in that the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus. 32. Genetically modified T cell according to any one of claims 25, 30 and 31, characterized by the fact that the other part of Cβ is encoded by a nucleotide sequence that comprises less than four exons, less than three exons, less than two exons, an exon, or less than a complete exon the open reading frame of a TRBC locus. 33. Genetically modified T cell according to any one of claims 25 and 30 to 32, characterized by the fact that the other part of Cβ is encoded by a nucleotide sequence that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long. 34. Genetically modified T cell according to any one of claims 25 and 30 to 33, characterized by the fact that the other part of Cβ is encoded by an exon 1 part of a TRBC locus, wherein the exon 1 part is smaller than the natural size of exon 1 of the open reading frame of the TRBC locus. 35. Genetically modified T cell according to any one of claims 24 to 34, characterized by the fact that the transgene sequence has been integrated downstream of the more 5 'exon 1 nucleotide and upstream of the more 3' exon nucleotide 1 of the open reading structure of the endogenous TRBC locus. 36. Genetically modified T cell according to any one of claims 24 to 35, characterized in that said at least one part of Cβ is encoded by at least one part of exon 1 and exons 2 to 4 of the structure of open reading of the endogenous TRBC locus. 37. Genetically modified T cell according to any one of claims 24 to 36, characterized by the fact that the encoded TCRβ chain is able to dimerize with the TCRα chain. 38. Genetically modified T cell according to any one of claims 24 to 37, characterized in that the encoded Cβ comprises the sequence selected from any one of SEQ ID NO: 16, 17, 21, and 25, or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity sequence with any one of SEQ ID NO: 16, 17, 21, and 25, or a partial sequence thereof. 39. Genetically modified T cell according to any one of claims 24 to 38, characterized in that the encoded Cβ comprises the sequence selected from any one of SEQ ID NO: 20, 21, 25 and 253 to 258 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity sequence with any one of SEQ ID NO: 20, 21, 25 and 253 to 258, or a partial sequence thereof. 40. Genetically modified T cell according to any one of claims 25 and 30 to 39, characterized by the fact that said at least part of Cβ is encoded by: a nucleotide sequence starting from residue 3 and up to residue 1445 of sequence mentioned in SEQ ID NO: 2 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons of same, or a partial sequence of them; or a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof, or a partial sequence thereof. 41. Genetically modified T cell according to any one of claims 25 and 30 to 39, characterized in that the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications , optionally a substitution, deletion, or insertion of one or more amino acids in comparison to a native Cβ region and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. 42. Genetically modified T cell according to claim 25 or 41, characterized by the fact that the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. 43. Genetically modified T cell according to any one of claims 25, 41 and 42, characterized by the fact that a coded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO: 20. 44. Genetically modified T cell according to any one of claims 25 and 41 to 43, characterized by the fact that the encoded Cβ comprises the sequence selected from any one of SEQ ID NOS: 253 and 256 to 258, or a sequence part of them. 45. Genetically modified T cell according to any one of claims 24 to 44, characterized in that the modified T cell also comprises a genetic disruption at a TRAC locus. 46. Genetically modified T cell according to any one of claims 1 to 45, characterized by the fact that the transgene sequence comprises one or more multicistronic elements. 47. Genetically modified T cell according to claim 46, characterized by the fact that the multicistronic elements are positioned between the nucleic acid sequence that co-complicates TCRα or a part of it and the nucleic acid sequence that encodes TCRβ or a part of it and / or upstream of the nucleic acid sequence encoding TCR or a part of TCR. 48. Genetically modified T cell according to claim 46 or 47, characterized by the fact that the multistronic element is or comprises a ribosome jump sequence, optionally T2A, P2A, E2A, or F2A. 49. Genetically modified T cell according to any one of claims 1 to 48, characterized in that the transgene sequence comprises one or more heterologous or regulatory control elements operably linked to control TCR expression when expressed from one cell introduced with the genetically modified T cell. 50. Genetically modified T cell according to claim 49, characterized by the fact that the heterologous or control regulatory element comprises a heterologous promoter. 51. Genetically modified T cell according to claim 50, characterized in that the heterologous promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and / or a tissue specific promoter. 52. A genetically modified T cell according to claim 50 or 51, characterized in that the heterologous promoter is or comprises a human elongation factor 1 alpha promoter or an MND promoter or a variant thereof. 53. T cell genetically modified according to any one of claims 1 to 52, characterized by the fact that the recombinant TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition. 54. Genetically modified T cell according to claim 53, characterized by the fact that the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease mune, an inflammatory disease, a tumor, or a cancer. 55. Genetically modified T cell according to any one of claims 1 to 54, characterized in that the T cell is a primary T cell derived from an individual, optionally in which the individual is a human. 56. Genetically modified T cell according to any one of claims 1 to 55, characterized in that the T cell is a CD8 + T cell or subtypes thereof. 57. Genetically modified cell according to any one of claims 1 to 55, characterized by the fact that the T cell is a CD4 + T cell or subtypes thereof. 58. Genetically modified cell according to any one of claims 1 to 57, characterized by the fact that the T cell is derived from a multipotent or pluripotent cell, which optionally is an iPSC. 59. Composition, characterized by the fact that it comprises a plurality of genetically modified T cells as defined in any one of claims 1 to 58. 60. Composition, characterized by the fact that it comprises a plurality of genetically modified T cells that comprise a modified alpha T cell receptor (TRAC) constant locus, said modified TRAC locus that comprises a nucleic acid encoding a Recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) an alpha TCR chain (TCRα) comprising a variable alpha domain (Vα) and a constant alpha domain (Cα), and ( ii) a beta TCR (TCRβ) chain comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoded by the TCRβ and the Vα, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, where the open reading frame encodes at least a part of the recombinant TCR Cα domain, and said recombinant TCR is able to bind to an antigen that is associated associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition; where: at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a T cell receptor beta constant region (TRBC) gene; at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express or do not express detectable levels of a gene product from an endogenous TRAC or TRBC gene; and / or at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the Recombinant TCR and / or exhibit antigen binding. 61. Composition comprising a plurality of genetically modified T cells, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, said modified TRAC locus comprising a nucleic acid encoding a Recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) an alpha TCR chain (TCRα) comprising a variable alpha domain (Vα) and a constant alpha domain (Cα), and ( ii) a beta TCR (TCRβ) chain comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), wherein the nucleic acid sequence comprises (a) a transgene sequence encoded by the TCRβ and the Vα, and (b) and an open reading frame of the endogenous TRAC locus or a partial sequence thereof, wherein the open reading frame encodes at least part of the recombinant TCR Cα domain; a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, where said other part of Cα is smaller than the size native to a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, referred to one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. 62. Composition, characterized by the fact that it comprises a plurality of genetically modified T cells that comprise a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus that comprises a nucleic acid which encodes a recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the nucleic acid sequence comprises (a) a transgene sequence encoding the TCRα and the Vβ domain, and (b) and an open reading frame of the endogenous TRBC locus or a partial sequence thereof, where the open reading frame encodes at least a part of the Cβ domain of the TCR recombinant, and said recombinant TCR is able to bind to an antigen that and is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition; where: at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a T cell receptor beta constant region (TRBC) gene; at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express or do not express detectable levels of a gene product from an endogenous TRAC or TRBC gene; and / or at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, or 90% of the cells in the composition express the Recombinant TCR and / or exhibit antigen binding. 63. Composition, characterized by the fact that it comprises a plurality of genetically modified T cells that comprise a modified T cell receptor (TRBC) beta constant locus, said modified TRBC locus that comprises a nucleic acid which encodes a recombinant TCR or part thereof, said recombinant TCR or part thereof, comprising: (i) a beta TCR chain (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ), and (ii) an alpha TCR chain (TCRα) comprising an alpha variable domain (Vα) and an alpha constant domain (Cα), where the nucleic acid sequence comprises (a) a transgene sequence encoding the TCRα and the Vβ domain, and (b) and an open reading frame of the endogenous TRBC locus or a partial sequence thereof, where the open reading frame encodes at least a part of the Cβ domain of the TCR recombinant; where: a part of the Cβ is encoded by the open reading structure part of the endogenous TRBC locus or a partial sequence thereof, and another part of the Cβ is encoded by the transgene sequence, wherein said other part of Cβ is smaller than the natural size of a native Cβ; and the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally a substitution, deletion, or insertion of one or more amino acids compared to a region Native Cβ and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain. 64. Composition according to claim 61 or 63, characterized by the fact that the recombinant TCR is capable of binding to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that it is associated with a disease, disorder, or condition. 65. Composition according to any one of claims 59 to 64, characterized in that the composition comprises CD4 + and / or CD8 + T cells. 66. Composition according to any one of claims 59 to 65, characterized in that the composition comprises CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is from or about 1: 3 to 3: 1, optionally 1: 1. 67. Composition according to any one of claims 59, 61 and 63 to 66, characterized by the fact that: at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a beta T cell receptor (TRBC) constant region; and / or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express or do not express detectable levels of an endogenous TRAC or TRBC gene product. 68. Composition according to any one of claims 59, 61 and 63 to 67, characterized by the fact that at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65 %, 70%, 75%, 80%, or 90% of the cells in the composition express the recombinant TCR and / or exhibit antigen binding. 69. Polynucleotide, characterized by the fact that it comprises: (a) a nucleic acid sequence encoded a part of a recombinant T cell receptor (TCR), said nucleic acid sequence encoding (i) a beta receptor chain T cell (TCRβ) comprising a variable beta domain (Vβ) and a constant beta domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is smaller than a natural size of a TCRα chain, and (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homologous arms comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus; in which the polynucleotide is comprised of a viral vector. 70. Polynucleotide, characterized by the fact that it comprises: (a) a nucleic acid sequence encoded a part of a recombinant T cell receptor (TCR), said nucleic acid sequence encoding (i) a beta receptor chain T cell (TCRβ) that comprises a variable beta domain (Vβ) and a constant beta domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is smaller than a natural size of a TCRα chain, and (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homologous arms comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus; where, when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide: a part of Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, wherein said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, referred to one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more more non-native cysteines. 71. Polynucleotide, characterized by the fact that it comprises: (a) a nucleic acid sequence encoded a part of a recombinant T cell receptor (TCR), said nucleic acid sequence encoding (i) a beta receptor chain T cell (TCRβ) comprising a beta variable domain (Vβ) and a beta constant domain (Cβ); and (ii) a part of an alpha T cell receptor (TCRα) chain, where the part of the TCRα chain is smaller than a natural size of a TCRα chain, and (b) one or more homology arms linked to the nucleic acid sequence, wherein said one or more homologous arms comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus; wherein the transgene sequence comprises one or more heterologous or regulatory control elements comprising a heterologous promoter, operably linked to control TCR expression when expressed from a cell introduced with the genetically modified T cell - gives. 72. Polynucleotide according to any one of claims 69 to 71, characterized by the fact that the TCRα chain comprises a constant alpha region (Cα), in which at least a part of said Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide; and / or the nucleic acid sequence of (a) and the aforementioned one or more of the homology arms together comprise a nucleotide sequence encoding Cα that is smaller than the natural size of a Native Cα, in which at least a part of Cα is encoded by the open reading frame of the endogenous TRAC locus or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide. 73. Polynucleotide according to any one of claims 69 to 72, characterized in that the nucleic acid sequence encoding the TCRβ chain is upstream of the nucleic acid sequence encoding a part of the TCRα chain. 74. Polynucleotide according to any one of the vindications 69 to 73, characterized by the fact that the nucleic acid sequence of (a) does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron. 75. Polynucleotide according to any of claims 69 to 74, characterized by the fact that the nucleic acid sequence of (a) is in structure with one or more exons or a partial sequence thereof of the reading structure of the TRAC locus comprised in said one or more homology arms. 76. Polynucleotide according to any of claims 69 and 71 to 75, characterized by the fact that a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of another part Cα is encoded by the nucleic acid sequence of (a), where said other part of Cα is smaller than the natural size of a native Cα. 77. Polynucleotide according to any one of claims 69 to 76, characterized by the fact that the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRAC locus. 78. Polynucleotide according to any one of claims 70, 76 and 77, characterized by the fact that the other part of Cα is encoded by a nucleotide sequence that comprises less than four exons, less than three exons , less than two exons, one exon, or less than a full exon of the open reading structure of the TRAC locus. 79. Polynucleotide according to any of claims 70 and 76 to 78, characterized by the fact that the other part of Cα is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long. 80. Polynucleotide according to any of claims 70 and 76 to 79, characterized by the fact that the other part of Cα is encoded by an exon 1 part of the TRAC locus, in which the exon 1 part is smaller than than the natural size of exon 1 of the open reading structure of the TRAC locus. 81. Polynucleotide according to any one of claims 69 to 80, characterized by the fact that the TCRα chain is able to dimerize with a TCRβ chain, when produced from a cell introduced with the polynucleotide. 82. Polynucleotide according to any of claims 69 to 81, characterized by the fact that the encoded Cα comprises the sequence selected from any of SEQ ID NOS: 19, 24 and 243 to 252, or a sequence that displays at least minus 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NOS: 19, 24 and 243 to 252, or a partial sequence thereof, when produced from a cell introduced with the polynucleotide. 83. Polynucleotide according to any one of claims 70 and 76 to 82, characterized by the fact that said at least one part of Cα is encoded by a sequence of nucleotides starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of the same or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 3155 of the sequence mentioned in SEQ ID NO: 1 or one or more exons of it, or a partial sequence of it. 84. Polynucleotide according to any of claims 70 and 76 to 83, characterized by the fact that the other part of Cα comprises a sequence mentioned in SEQ ID NO: 142, or a sequence that exhibits at least 85%, 86 %, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with SEQ ID NO: 142, or a partial sequence thereof. 85. Polynucleotide according to any of claims 76 to 84, characterized by the fact that the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one substitution, deletion, or insertion of one or more amino acids in comparison to a native Cα region and / or a native Cβ region, optionally said one or more modifications introduce one or more cysteine residues that are capable of form one or more non-native disulfide bridges between the alpha chain and the beta chain. 86. Polynucleotide according to claims 70 and 85, characterized by the fact that the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. 87. Polynucleotide according to any of claims 70, 85 and 86, characterized by the fact that a coded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO : 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO: 20, when produced from a cell introduced with the polynucleotide. 88. Polynucleotide according to any of claims 70 and 85 to 87, characterized by the fact that the encoded Cα comprises the selected sequence of any one of SEQ ID NOS: 248-252, or a partial sequence thereof , when from a cell introduced with the polynucleotide. 89. Polynucleotide according to any of claims 69 to 88, characterized by the fact that the part of the TCRα chain comprises a variable alpha domain (Vα). 90. Polynucleotide according to any one of claims 69 to 89, characterized in that said one or more homology arms comprises a 5 'homology arm and / or a 3' homology arm. 91. Polynucleotide according to claim 90, characterized by the fact that the 5 'homology arm and the 3' homology arm comprise nucleic acid sequences homologous to the nucleic acid sequences that surround a target site, in that the target site is within the TRAC locus. 92. Polynucleotide according to claim 91, characterized by the fact that the target site is within exon 1 of the TRAC locus. 93. Polynucleotide according to any one of claims 90 to 92, characterized by the fact that the 5 'homology arm comprises: a) a sequence comprising, or comprising at least 150, 200, 250, 300 , 350, 400, 450, 500, 550, or 600 nucleotides contiguous to a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or more of sequence identity with the sequence mentioned in SEQ ID NO: 124; b) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence mentioned in SEQ ID NO: 124; oU c) the sequence mentioned in SEQ ID NO: 124. 94. Polynucleotide according to any one of the vindications 90 to 93, characterized by the fact that the homology arm 3 'comprises: a) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 nucleotides contiguous to a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or more of sequence identity with the sequence mentioned in SEQ ID NO: 125; b) a sequence comprising, or comprising at least 150, 200, 250, 300, 350, 400, 450, 500, 550, or 600 contiguous nucleotides of the sequence mentioned in SEQ ID NO: 125; or c) the sequence mentioned in SEQ ID NO: 125. 95. Polynucleotide, characterized by the fact that it comprises: (a) a nucleic acid sequence encoded a part of a recombinant T cell receptor (TCR), said nucleic acid sequence that encodes (i) an alpha chain of T cell receptor (TCRα) comprising a variable alpha domain (Vα) and a constant alpha domain (Cα); and (ii) a part of a beta T cell receptor (TCRβ) chain, where the part of the TCRβ chain is smaller than a native, full-size TCRβ chain, and (b) one or more homology arms attached to the nucleic acid sequence, wherein said one or more homologous arms comprise a sequence homologous to one or more regions of an open reading frame of a TRBC locus. 96. Polynucleotide according to claim 95, characterized by the fact that the TCRβ chain comprises a constant beta (Cβ), in which at least a part of said Cβ is encoded by the open reading structure of the locus Endogenous TRAC or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide and / or the nucleic acid sequence of (a) and the cited one of those one or more homology arms together comprise a nucleotide sequence encoding Cβ that is smaller than the natural size of a native Cβ, in which at least part of the Cβ is encoded by the open reading structure of the locus Endogenous TRBC or a partial sequence thereof when the TCR or antigen-binding fragment thereof is expressed from a cell introduced with the polynucleotide. 97. Polynucleotide according to claim 95 or 96, characterized in that the TRBC locus is one or more of TRBC1 or TRBC2. 98. Polynucleotide according to any one of claims 95 to 97, characterized by the fact that the nucleic acid sequence encoding the TCRα chain is upstream of the nucleic acid sequence encoding the part of the TCRβ chain. 99. Polynucleotide according to any of claims 95 to 98, characterized by the fact that the nucleic acid sequence of (a) does not comprise a sequence encoding a 3 'untranslated region (3' UTR) or an intron. 100. Polynucleotide according to any of claims 95 to 99, characterized by the fact that the nucleic acid sequence of (a) is in structure with one or more exons or a partial sequence thereof of the reading structure of the TRAC locus comprised in said one or more homology arms. 101. Polynucleotide according to any of claims 95 to 100, characterized by the fact that a part of the Cβ is encoded by the open reading structure of the endogenous TRBC locus or a partial sequence thereof, and another part of the Cβ is encoded by the nucleic acid sequence of (a), where said other part of Cβ is smaller than the natural size of a native Cβ 102. Polynucleotide according to any of claims 95 to 101, characterized by the fact that the open reading frame or the partial sequence thereof comprises at least one intron and at least one exon of the endogenous TRBC locus. 103. Polynucleotide according to claim 101 or 102, characterized in that the other part of Cβ is encoded by a nucleotide sequence that encodes less than four exons, less than three exons, less than two exons, one exon, or less than a full exon of the open reading frame of the TRBC locus. 104. Polynucleotide according to any of claims 101 to 103, characterized by the fact that the other part of Cβ is encoded by a sequence of nucleotides that is less than 400, less than 300, less than 250, less than 200, less than 150, base pairs long. 105. Polynucleotide according to any of claims 101 to 104, characterized by the fact that the other part of Cβ is encoded by an exon 1 part of a TRBC locus, in which the exon 1 part is less than the natural size of exon 1 of the open reading structure of the TRBC locus. 106. Polynucleotide according to any of claims 95 to 105, characterized by the fact that the TCRβ chain is able to dimerize with a TCRα chain, when produced from a cell introduced with the polynucleotide. 107. Polynucleotide according to any one of claims 69 to 106, characterized by the fact that the encoded Cβ comprises the sequence selected from any of SEQ ID NO: 20, 21, 25 and 253-258 or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with any of SEQ ID NO: 20, 21, 25 and 253-258, or a partial sequence thereof, when produced from a cell introduced with the polynucleotide. 108. Polynucleotide according to any of claims 101 to 107, characterized by the fact that said at least a part of Cβ is encoded by: a sequence of nucleotides starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons of the same or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1445 of the sequence mentioned in SEQ ID NO: 2 or one or more exons thereof, or a partial sequence of them; or a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof or a sequence that exhibits at least 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a nucleotide sequence starting from residue 3 and up to residue 1486 of the sequence mentioned in SEQ ID NO: 3 or one or more exons thereof, or a partial sequence thereof. 109. Polynucleotide according to any of claims 101 to 108, characterized by the fact that the other part of the Cβ and / or the Cα region encoded by the nucleic acid sequence of (a) comprises one or more modifications, optionally one substitution, deletion, or insertion of one or more amino acids in comparison with a native Cβ region and / or a native Cα region, optionally said one or more modifications introduce one or more cysteine residues that are capable of form one or more non-native disulfide bridges between the alpha chain and the beta chain. 110. Polynucleotide according to claim 109, characterized in that the introduction of one or more cysteine residues comprises the replacement of a non-cysteine residue with a cysteine residue. 111. Polynucleotide according to claim 109 or 110, characterized in that an encoded Cα region comprises a cysteine in a position that corresponds to position 48 with numbering as mentioned in any of SEQ ID NO: 24; and / or the coded Cβ region comprises a cysteine in a position that corresponds to position 57 with numbering as mentioned in SEQ ID NO: 20. 112. Polynucleotide according to any one of claims 109 to 111, characterized by the fact that the encoded Cβ comprises the selected sequence of any one of SEQ ID NOS: 253 and 256-258, or a partial sequence thereof. 113. Polynucleotide according to any one of claims 95 to 112, characterized by the fact that the part of the TCRβ chain comprises a variable beta domain (Vβ). 114. Polynucleotide according to any one of claims 95 to 113, characterized in that said one or more homology arms comprises a 5 'homology arm and / or a 3' homology arm. 115. Polynucleotide according to claim 114, characterized in that the 5 'homology arm and the 3' homology arm comprise nucleic acid sequences homologous to the nucleic acid sequences surrounding a target site, wherein the target site is within the open reading structure of the TRBC locus. 116. Polynucleotide according to claim 115, characterized by the fact that the target site is within exon 1 of the open reading frame of the TRBC locus. 117. Polynucleotide according to any of claims 69 to 116, characterized by the fact that the nucleic acid sequence of (a) is a sequence that is exogenous or heterologous to an open reading frame of an endogenous genomic TRAC locus - that of a T cell, optionally a human T cell. 118. Polynucleotide according to any of claims 90 to 94 and 114 to 117, characterized by the fact that the 5 'homology arm and the 3' homology arm independently are between 50 or about and 100 or about of, nucleotides in length, 100 or about, and 250 or about, nucleotides in length, 250 or about, and 500 or about, nucleotides in length, 500 or about, and 750 or about, nucleotides in length , 750 or about, and 1,000 or about, nucleotides in length, 1,000 or about, and 2,000 or about, nucleotides in length. 119. Polynucleotide according to any one of claims 90 to 94 and 114 to 118, characterized by the fact that the 5 'homology arm and the 3' homology arm independently are either about 100 to 1,000 or about, nucleotides, 100 to 750 nucleotides, 100 to 600 nucleotides, 100 to 400 nucleotides, 100 to 300 nucleotides, 100 to 200 nucleotides, 200 to 1000 nucleotides, 200 to 750 nucleotides, 200 to 600 nucleotides, 200 to 400 nucleotides, 200 to 300 nucleotides, 300 to 1000 nucleotides, 300 to 750 nucleotides, 300 to 600 nucleotides, 300 to 400 nucleotides, 400 to 1000 nucleotides, 400 to 750 nucleotides, 400 to 600 nucleotides, 600 to 1000 nucleotides , 600 to 750 nucleotides or 750 to 1000 nucleotides in length. 120. Polynucleotide according to any of claims 90 to 94 and 114 to 119, characterized by the fact that the Brazilian homology section 5 'and the homology arm 3' independently are either about 200, 300, 400, 500, 600, 700 or 800 nucleotides in length, or any value between any of the above. 121. Polynucleotide according to any of claims 90 to 94 and 114 to 120, characterized by the fact that the 5 'homology arm and the 3' homology arm independently are greater than or about 300 nucleotides in length, optionally wherein the 5 'homology arm and the 3' homology arm independently are either about 400, 500 or 600 nucleotides in length or any value between any of the foregoing. 122. Polynucleotide according to any of claims 90 to 94 and 114 to 121, characterized by the fact that the 5 'homology arm and the 3' homology arm independently are, are about, or are of less than about 600 nucleotides in length. 123. Polynucleotide according to any of claims 90 to 94 and 114 to 122, characterized by the fact that the 5 'homology arm and the 3' homology arm are independently greater than or about 300 nucleotides of lenght. 124. Polynucleotide according to any one of claims 69 to 123, characterized by the fact that the nucleic acid sequence of (a) comprises one or more multicistronic elements. 125. Polynucleotide according to claim 124, characterized in that the multicistronic elements are positioned between the nucleic acid sequence that encodes TCRα or a part of it and the nucleic acid sequence that encodes TCRβ or a part and / or are upstream of the nucleic acid sequence encoding the TCR or a part of the TCR. 126. Polynucleotide according to claim 124 or 125, characterized by the fact that the multicistronic element is or comprises a ribosome jump sequence, optionally T2A, P2A, E2A, or F2A. 127. Polynucleotide according to any one of claims 69 to 126, characterized by the fact that the nucleic acid sequence of (a) comprises one or more heterologous or regulatory control elements operably linked to control TCR expression when expressed of a cell introduced with the polynucleotide. 128. Polynucleotide according to claim 127, characterized in that the heterologous or control regulatory element comprises a heterologous promoter. 129. Polynucleotide according to claim 128, characterized in that the heterologous promoter is selected from a constitutive promoter, an inducible promoter, a repressible promoter, and / or a tissue specific promoter. 130. Polynucleotide according to claim 128 or 129, characterized in that the heterologous promoter is or comprises a human elongation factor 1 alpha promoter or an MND promoter or a variant thereof. 131. Polynucleotide according to any of claims 69 to 130, characterized by the fact that the polynucleotide is comprised of a viral vector. 132. Polynucleotide according to claim 131, characterized by the fact that the viral vector is an AAV vector, optionally selected from the AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8 vector. 133. Polynucleotide according to claim 132, characterized in that the AAV vector is an AAV2 or AAV6 vector. 134. Polynucleotide according to any one of the vindications 69 to 133, characterized by the fact that the polynucleotide comprises the structure: [5 'homology arm] - [nucleic acid sequence of (a)] - [3' homology arm]. 135. Polynucleotide according to any of claims 124 to 134, characterized by the fact that the polynucleotide comprises the structure: [5 'homology arm] - [multi-cistronic element] - [nucleic acid sequence of (a)] - [3 'homology arm]. 136. Polynucleotide according to any of claims 128 to 134, characterized by the fact that the polynucleotide comprises the structure: [5 'homology arm] - [heterologous promoter] - [nucleic acid sequence of (a)] - [3 'homology arm]. 137. Polynucleotide according to any of claims 69 to 136, characterized by the fact that the recombinant encoded TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that is associated with a disease, disorder, or condition. 138. Polynucleotide according to claim 137, characterized in that the disease, disorder, or condition is an infectious disease or disorder, an autoimmune disease, an inflammatory disease, a tumor, or a cancer. 139. Polynucleotide according to any of claims 69 to 138, characterized by the fact that the polynucleotide is at least about or about 2,500, 2,750, 3,000, 3,250, 3,500, 3,750, 4,000, 4,250, 4,500, 4,760, 5,000, 5,250, 5,500, 5,750, 6,000, 7,000, 7,500, 8,000, 9,000 or 10,000 nucleotides in length, or any value between any of the above. 140. Polynucleotide according to any one of claims 69 to 139, characterized by the fact that the polynucleotide is between or about 2,500, and of or about 5,000 nucleotides, of or about 3,500 and of or about 4,500 nucleotides, or about 3,750 nucleotides and about or 4,250 nucleotides in length. 141. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified TRAC locus, comprising introducing the polynucleotide as defined in any of claims 69 to 94 or 117 to 140 into a T cell comprising a genetic disruption in a TRAC locus. 142. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, the method comprising: (a) introducing into one T cell, one or more agents capable of inducing a genetic disruption at a target site within an endogenous T cell TRAC site; and (b) introducing into the T cell a polyucleotide comprising a transgene sequence encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), wherein the part is smaller than a natural-sized TCRα chain, and in which: the introduction of the standard polynucleotide is carried out after the introduction of said one or more agents capable of inducing a genetic disruption; and the transgene is targeted for integration within the endogenous TRAC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRAC locus. 143. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, the method of introducing into a T cell a polynucleotide comprising a sequence of transgene encoding a T cell receptor beta chain (TCRβ) and part of a T cell receptor alpha chain (TCRα), said cell having a genetic disruption within the endogenous T cell TRAC locus, wherein the trans - gene is targeted for integration within the endogenous TRAC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRAC locus. 144. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, the method comprising: (a) introducing into one T cell, one or more agents capable of inducing a genetic disruption at a target site within an endogenous T cell TRAC site; and (b) introducing into the T cell a polyucleotide comprising a transgene sequence encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), wherein the part is smaller than a full-size native TCRα chain; and the transgene is directed towards integration within the endogenous TRAC locus through homology-directed repair (HDR); thereby producing a genetically modified cell comprising a modified TRAC locus, in which, after targeted integration of the transgene: a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, in which the other part of Cα is smaller than the natural size of a Native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, referred to one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more more non-native cysteines. 145. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified alpha T cell receptor (TRAC) constant locus, the method of introducing into a T cell a polynucleotide that comprises a sequence of transgene encoding a T cell receptor beta chain (TCRβ) and a part of a T cell receptor alpha chain (TCRα), said cell having a genetic disruption within the endogenous T cell TRAC locus; and the transgene is directed towards integration within the endogenous TRAC locus through homology-directed repair (HDR); thereby producing a genetically modified cell comprising a modified TRAC locus, in which, after targeted integration of the transgene: a part of Cα is encoded by the open reading structure of the endogenous TRAC locus or a partial sequence thereof, and another part of Cα is encoded by the transgene sequence, where said other part of Cα is smaller than the natural size of a native Cα; and the other part of Cα and / or the Cβ region encoded by the nucleic acid sequence of (a) comprises one or more modifications in comparison to a native Cα region and / or a native Cβ region, referred to one or more modifications introduce one or more cysteine residues that are capable of forming one or more non-native disulfide bridges between the alpha chain and the beta chain and / or in which the Cα and / or Cβ of the recombinant TCR comprises one or more more non-native cysteines. 146. Method according to any of claims 141, 143 and 145, characterized in that the genetic disruption was induced by one or more agents capable of inducing a genetic disruption of one or more target sites within the endogenous TRAC locus . 147. Method according to any one of claims 141 to 146, characterized in that the polynucleotide is polynucleotide as defined in any of claims 69 to 94 or 117 to 140. 148. Method according to any of claims 141 to 147, characterized in that the modified TRAC locus comprises a nucleic acid sequence encoding a recombinant TCR or part of it, wherein the nucleic acid sequence comprises a structure fusion of (i) a transgene sequence and (ii) and an open reading frame or partial sequence thereof of the endogenous TRAC locus, optionally wherein the transgene sequence is in structure with one or more exons of the open reading structure or partial sequence of the same of the endogenous TRAC locus. 149. Method according to any one of claims 141 to 148, characterized in that the modified T cell also comprises inducing a genetic disruption in a TRBC locus. 150. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified TRBC locus, comprising introducing the polynucleotide according to any one of claims 95 to 140 into a T cell that comprises a genetic disruption in a TRBC locus. 151. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified T cell receptor (TRBC) beta constant locus, the method comprising: (a) introducing, in a T cell, one or more agents capable of inducing a genetic disruption at a target site within an endogenous T cell locus TRBC; and (b) introducing into the T cell a polyucleotide comprising a transgene sequence encoding a T cell receptor alpha chain (TCRα) and a part of a T cell receptor beta chain (TCRβ), wherein the part is smaller than a native, full-size TCRβ chain, and in which the transgene is targeted for integration into an endogenous TRBC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRBC locus - of. 152. Method of producing a genetically modified T cell, characterized by the fact that it comprises a modified T cell receptor (TRBC) beta constant locus, the method of introducing into a T cell a polynucleotide that comprises a sequence of transgene encoding a T cell receptor alpha chain (TCRα) and a part of a T cell receptor beta chain (TCRβ), said cell having a genetic disruption within an endogenous T cell locus TRBC, wherein the transgene is targeted for integration within the endogenous TRBC locus by means of homology-directed repair (HDR), thereby producing a genetically modified cell that comprises a modified TRBC locus. 153. Method according to claims 150 and 152, characterized by the fact that the genetic disruption was induced by one or more agents capable of inducing a genetic disruption of one or more target sites within the endogenous TRBC locus. 154. Method according to any of claims 150 to 153, characterized in that the TRBC locus is a TRBC1 locus and / or a TRBC2 locus. 155. Method according to any one of claims 151 to 154, characterized in that the polynucleotide is polynucleotide according to any one of claims 95 to 140. 156. Method according to any one of claims 151 to 155, characterized in that the modified TRBC locus comprises a nucleic acid sequence encoding a recombinant TCR or part thereof, wherein the nucleic acid sequence comprises a structure fusion of (i) a transgene sequence and (ii) and an open reading frame or partial sequence thereof of the endogenous TRBC locus, optionally wherein the transgene sequence is in structure with one or more exons of the structure of open reading or partial sequence of the same of the endogenous TRBC locus. 157. Method according to any one of claims 151 to 156, characterized in that the modified T cell also comprises inducing a genetic disruption in a TRAC locus. 158. Method according to any of claims 142 and 144 to 157, characterized in that said one or more agents capable of inducing a genetic disruption comprises a DNA-binding protein or DNA-binding nucleic acid that specifically binds to or hybridizes to the target site. 159. Method according to any one of the claims 142 and 144 to 158, characterized by the fact that said one or more agents comprises a finger zinc protein (ZFP), a TAL protein, or a nease associated with short palindromic nucleic acid regularly interspersed in a cluster (CRISPR) (Cas) specific to the target site. 160. Method according to any one of claims 142 and 144 to 159, characterized in that said one or more agents comprise a combination of CRISPR-Cas9 and the combination of CRISPR-Cas9 a guide RNA (gRNA) having a targeting domain that is complementary to at least one target site. 161. Method according to claim 160, characterized in that said one or more agents are introduced as a ribonucleoprotein complex (RNP) comprising the gRNA and a Cas9 protein. 162. Method according to claim 161, characterized by the fact that the concentration of RNP is, or is about 1 µM to or about 5 µM, optionally wherein the concentration of RNP is, or is about 2 µM. 163. Method according to claim 161 or 162, characterized by the fact that RNP is introduced by means of electroporation. 164. Method according to any of claims 160 to 163, characterized by the fact that the gRNA has a targeting domain that is complementary to a target site in a TRAC site and comprises a sequence selected from the group consisting of UCUCUCAGCUGGUACACGGC (SEQ ID NO: 28), UG- GAUUUAGAGUCUCUCAGAGC (SEQ ID NO: 29), ACACGGCAGGGU- CAGGGUUC (SEQ ID NO: 30), GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31) ), CUCA- GCUGGUACACGGC (SEQ ID NO: 33), UGGUACACGGCAGGGUC (SEQ ID NO: 34), GCUAGACAUGAGGUCUA (SEQ ID NO: 35), GU- CAGAUUUGUUGCUCC (SEQ ID NO: 36), UCAGCUGGUACACGGAC (SEQ ID: 37) ID NO: 38), GGUACACGGCAGGGUCA (SEQ ID NO: 39), CUUCAAGAGCAACA- GUGCUG (SEQ ID NO: 40), AGAGCAACAGUGCUGUGGCC (SEQ ID NO: 41), AAAGUCAGAUUUGUUGCUCC (SEQ ID NO: 42), ACAAAAAAAAAAAAAA NO: 43), AAACUGUGCUAGACAUG (SEQ ID NO: 44), UGUGCUAGACAUGAGGUCUA (SEQ ID NO: 45), GGCUGGGGAAGAAGGUGUCUUC (SEQ ID NO: 46), GCUGGGGA- AGAAGGUGUCUUC (SEQ ID NO: 47), GAGGGA ), GUUUUGUCUGUGAUAUACACAU (SEQ ID NO: 49), GGCAGACAGACUUGUCACUGGAUU (SEQ ID NO: 50), GCAGACA- GACUUGUCACUGGAUU (SEQ ID NO: 51), GACAGACUUGUCA- CUGGAUU (SEQ ID NO: 52), GAGA , GAAUAGGCAGACAGACUUGUCA (SEQ ID NO: 54), GAGUCUCUCAGCUGGUACACGG (SEQ ID NO: 55), GUCUCUCAG- CUGGUACACGG (SEQ ID NO: 56), GGUACACGGCAGGGUCA- GGGUU (SEQ ID NO: 57) 165. Method according to claim 164, characterized by the fact that the gRNA has a targeting domain that comprises the sequence GAGAAUCAAAAUCGGUGAAU (SEQ ID NO: 31). 166. Method according to any of claims 160 to 163, characterized in that the gRNA has a targeting domain that is complementary to a target site in one or both of a TRBC1 and a TRBC2 gene and comprises a sequence selected from the group consisting of CACCCAGAUCGUCAG- CGCCG (SEQ ID NO: 59), CAAACACAGCGACCUCGGGU (SEQ ID NO: 60), UGACGAGUGGACCCAGGAUA (SEQ ID NO: 61), GGCU- CUCGGAGAAUGACGAG (SEQ ID NO: 62), GGCCUCGGCGCUGA- CGAUCU (SEQ ID NO: 63), GAAAAACGUGUUCCCACCCG (SEQ ID NO: 64) : 65), AGUCCA- GUUCUACGGGCUCU (SEQ ID NO: 66), CGCUGUCAAGUCCAGUU- CUA (SEQ ID NO: 67), AUCGUCAGCGCCGAGGCCUG (SEQ ID NO: 68), UCAAACACAGCGACCUCGG (SEQ ID NO: 69), SEAGGU 70), AGGCCUCGGCG- CUGACGAUC (SEQ ID NO: 71), UGACAGCGGAAGUGGUUGCG (SEQ ID NO: 72), UUGACAGCGGAAGUGGUUGC (SEQ ID NO: 73), UCUCCGAGAGCCCGUAGAAC (SEQ ID NO: 74), SEAGGU ), GACAGGUUUGGCCCUAUCCU (SEQ ID NO: 76), GAUCGUCAGCGCCGAGGCCU (SEQ ID NO: 77), GGCUCAAACACAGCGACCUC (SEQ ID NO: 78), UGAGGGU- CUCGGCCACCUUC (SEQ ID NO: 80), AGGU , CCGACCACGUGGAGCUGAGC (SEQ ID NO: 81), UGACAGGUUUGGCCCUAUCC (SEQ ID NO: 82), CUUGA- CAGCGGAAGUGGUUG (SEQ ID NO: 83), AGAUCGUCAGCG- CCGAGGCC (SEQ ID NO: 84), SEQGUGUGAC UGAGGGCGGGCUGCUCCUUG (SEQ ID NO: 86), GUUGCGGGGG UUCUGCCAGA (SEQ ID NO: 87), AGCUCAGCUC- CACGUGGUCG (SEQ ID NO: 88), GCGGCUGCUCAGGCAGUAUC (SEQ ID NO: 89), GCGGGGGUUCUGCCAGAAGG (SEQ ID NO: 90), UGGCUCAAACACGAGGAGACGACA (SEQ ID NO: 92), GACAGCGGAAGUGGUUGCGG (SEQ ID NO: 93), GCUGUCAAGUCCAGUUCUAC (SEQ ID NO: 94), GUAUCUGGAGUCAUUGAGGG (SEQ ID NO: 95), CUCGGCGCUGA- CGAUCU (SEQ ID NO: 96) NO: 97), CCGAGAGCCCGUAGAAC (SEQ ID NO: 98), CCAGAUCGU- CAGCGCCG (SEQ ID NO: 99), GAAUGACGAGUGGACCC (SEQ ID NO: 100), GGGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 101), GGUGACAGGUUUGGCCCUAUC (SEQ ID NO: 102), GUGACA- GGUUUGGCCCUAUC (SEQ ID NO: 103), GACAGGUUUGG- CCCUAUC (SEQ IDCC: 104) : 105), GACCACGUGGAGCUGAGCUGGUGG (SEQ ID NO: 106), GUGGAGCUGAGCUGGUGG (SEQ ID NO: 107), GGG- CGGGCUGCUCCUUGAGGGGCU (SEQ ID NO: 108), GGCGGG- CUGCUCCUUGGGGGGGGGGGGGGGGGGGGG NO: 110), GGGCUGCUCCUUGAGGGG- CU (SEQ ID NO: 111), GGCUGCUCCUUGAGGGGCU (SEQ ID NO: 112), GCUGCUCCUUGAGGGGCU (SEQ ID NO: 113), GGU- GAAUGGGAAGGAGGUGCACAGAGGAGGAGA ID NO: 115) and GAAUGGGAAGGA- GGUGCACAG (SEQ ID NO: 116). 167. Method according to claim 166, characterized by the fact that the gRNA has a targeting domain that comprises the sequence GGCCUCGGCGCUGACGAUCU (SEQ ID NO: 63). 168. Method according to any one of claims 141 to 167, characterized in that the T cell is a primary T cell of an individual. 169. Method according to claim 168, characterized by the fact that the individual has or is suspected of having the disease, disorder or condition. 170. Method according to claim 168, characterized by the fact that the individual is, or is suspected of being healthy. 171. Method according to claim according to any one of claims 141 to 170, characterized in that the T cell is a CD8 + T cell or subtypes thereof. 172. Method according to any one of the claims 141 to 170, characterized by the fact that the T cell is a CD4 + T cell or subtypes thereof. 173. Method according to any of claims 141 to 167, characterized in that the T cell is derived from a multipotent or pluripotent cell, which is optionally an iPSC. 174. Method according to any one of claims 168 to 173, characterized in that the T cell comprises a T cell that is autologous to the individual. 175. Method according to any of claims 168 to 173, characterized in that the T cell comprises a T cell that is allogeneic to the individual. 176. Method according to any of claims 141 to 175, characterized in that the polynucleotide is comprised of a viral vector. 177. Method according to claim 176, characterized by the fact that the vector is an AAV vector, optionally selected from vector AV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7 or AAV8. 178. Method according to claim 177, characterized by the fact that the AAV vector is an AAV2 or AAV6 vector. 179. Method according to any of claims 141 to 178, characterized by the fact that the recombinant TCR is able to bind to an antigen that is associated with, specific for, and / or expressed in a cell or tissue that it is associated with a disease, disorder, or condition. 180. Method according to any one of claims 144 to 179, characterized in that the introduction of said one or more agents capable of inducing a genetic disruption and an introduction of the standard polynucleotide are carried out simultaneously. sequentially or in any order. 181. Method according to any of claims 144 to 180, characterized by the fact that the introduction of the standard polynucleotide is carried out after the introduction of said one or more agents capable of inducing a genetic disruption. 182. Method according to claim 181, characterized in that the standard polynucleotide is introduced immediately after, or within or about 30 seconds, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 6 minutes, 8 minutes, 9 minutes, 10 minutes, 15 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours or 4 hours after the introduction of one or more agents capable of inducing a genetic disruption, optionally of or about 2 hours after the introduction of said one or more agents. 183. Method according to any one of claims 141 to 182, characterized in that the method is carried out on a plurality of T cells. 184. Method according to claim 183, characterized in that the plurality of T cells comprises CD4 + T cells, CD8 + T cells or CD4 + and CD8 + T cells. 185. Method according to claim 183 or 184, characterized in that the plurality of T cells comprise CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is about 1: 3 to about or 3: 1, optionally from or about 1: 2 to or about 2: 1, optionally from or about 1: 1. 186. Method according to any of claims 142 and 144 to 185, characterized by the fact that before the introduction of said one or more agents, the method comprises incubating the cells, in vitro with a stimulating agent under conditions to stimulate or activate said one or more T cells. 187. Method according to claim 186, characterized in that the stimulating agents comprise anti-CD3 and / or anti-CD28 antibodies, optionally anti-CD3 / anti-CD28 beads, optionally in which the ratio of cell count is, or is about 1: 1. 188. Method according to claim 186 or 187, characterized in that it comprises removing the stimulating agents from said one or more immune cells prior to introduction with said one or more agents. 189. Method according to any of claims 142 and 144 to 185, characterized in that the method also comprises incubating the cells before, during or subsequent to the introduction of said one or more agents and / or introduction of the standard polynucleotide with one or more recombinant cytokines, optionally wherein said one or more recombinant cytokines are selected from the group consisting of IL-2, IL-7, and IL-15. 190. The method of claim 189, characterized in that said one or more recombinant cytokines are added at a selected concentration of an IL-2 concentration from or about 10 U / ml to or about 200 U / ml, optionally to or about 50 IU / ml to or about 100 U / ml; IL-7 at a concentration of 0.5 ng / mL to 50 ng / mL, optionally from or about 5 ng / mL to or about 10 ng / mL and / or IL-15 at a concentration of 0 , 1 ng / ml to 20 ng / ml, optionally from or about 0.5 ng / ml to or about 5 ng / ml. 191. Method according to claim 189 or 190, characterized in that the incubation is carried out subsequent to the introduction of said one or more agents and the introduction of the standard polynucleotide for up to or approximately 24 hours, 36 hours, 48 hours , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 days, optionally up to or about 7 days. 192. Method according to any one of claims 141 to 191, characterized in that the polynucleotide is at least less than or about 2500, 2750, 3000, 3250, 3500, 3750, 4000, 4,250, 4500, 4760, 5000, 5250, 5500, 5750, 6000, 7000, 7500, 8000, 9000 or 10,000 nucleotides in length, or any value between any of the above. 193. Method according to any one of claims 141 to 192, characterized in that the polynucleotide is between or about 2,500, and of or about 5,000 nucleotides, of or about 3,500 and of or about of 4,500 nucleotides, or of or about 3,750 nucleotides and of or about 4,250 nucleotides in length. 194. Modified T cell or a plurality of modified T cells, characterized by the fact that they are generated using the method as defined in any of claims 141 to 193. 195. Composition, characterized by the fact that it comprises the modified T cell or a plurality of modified cells as defined in claim 194. 196. Composition according to claim 195, characterized by the fact that it comprises CD4 + and / or CD8 + T cells. 197. Composition according to claim 195 or 196, characterized in that the composition comprises CD4 + and CD8 + T cells and the ratio of CD4 + T cells to CD8 + is about 1: 3 to 3: 1, optionally 1 :1. 198. Composition according to any of claims 195 to 197, characterized by the fact that: at least 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97%, 98%, or 99% of the modified cells in the composition comprise a genetic disruption in or of an endogenous T cell receptor (TRC) alpha constant region gene and / or a beta constant region gene of T cell receptor (TRBC); and / or at least 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the modified cells in the composition do not express or do not express detectable levels of an endogenous TRAC or TRBC gene product. 199. Composition according to any of claims 195 to 198, characterized by the fact that at least or more than 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% , 75%, 80%, or 90% of the cells in the composition express the recombinant TCR and / or exhibit antigen binding. 200. Method of treatment, characterized by the fact that it comprises administering to the individual the modified cell, a plurality of modified cells or composition as defined in any one of claims 194 to 199. 201. Use of the modified cell, a plurality of modified cells or composition according to any one of claims 194 to 199, characterized by the fact that it is intended for the treatment of a disease or disorder. 202. Use of the modified cell, a plurality of modified cells or composition according to any one of claims 194 to 199, characterized by the fact that it is intended for the manufacture of a medicament for the treatment of a disease or disorder bio. 203. Modified cell, a plurality of modified cells or composition according to any one of claims 194 to 199 characterized by the fact that it is intended for use in the treatment of a disease or disorder. 204. Article of manufacture, characterized by the fact that it comprises: polynucleotide as defined in any of claims 69 to 94 or 117 to 140, and one or more agents capable of inducing a genetic disruption at a target site within a TRAC locus. 205. Article of manufacture, characterized by the fact that it comprises: a polynucleotide comprising (a) a nucleic acid sequence that encodes a beta T cell receptor (TCRβ) chain and part of an alpha T cell receptor chain (TCRα), where the part is smaller than a natural-sized native TCRα chain and (b) one or more homology arms linked to the nucleic acid sequence in which said one or more homology arms (s) comprise a sequence homologous to one or more regions of an open reading frame of a TRAC locus, the referred to open reading frame encoding a TCRα chain; and one or more agents capable of inducing a genetic disruption at a target site within a TRAC locus. 206. The article of manufacture according to claim 205, characterized in that the polynucleotide comprises polynucleotide as defined in any of claims 69 to 94 or 117 to 140. 207. Article of manufacture, characterized by the fact that it comprises: polynucleotide as defined in any one of claims 95 to 140, and one or more agents capable of inducing a genetic rupture at a target site within a TRBC locus. 208. Article of manufacture, characterized by the fact that it comprises: a polynucleotide comprising (a) a nucleic acid sequence that encodes an alpha T cell receptor chain (TCRα) and part of a beta T cell receptor chain (TCRβ), where the part is smaller than a natural-sized native TCRβ chain and (b) one or more homology arms linked to the nucleic acid sequence in which said one or more homology arms (s) comprise a sequence homologous to one or more regions of an open reading frame of a TRBC locus, the referred to open reading frame encoding a TCRβ chain; and one or more agents capable of inducing a genetic disruption at a target site within a TRAC locus. 209. Article of manufacture according to claim 207 or 208, characterized in that the polynucleotide comprises polynucleotide as defined in any of claims 95 to 140. 210. Kit, characterized by the fact that it comprises an article of manufacture as defined in any one of claims 204 to 209, and instructions for use.